Cancer treatments

ABSTRACT

Provided are medical uses and methods for targeting cancer stem cells employing ProTide compounds, particularly in the prevention or treatment of cancer. The ProTide may be other than one selected from the group consisting of: NUC-1031; a ProTide derived from cordycepin; and a ProTide derived from 8-chloroadenosine. The medical uses and methods for targeting cancer stem cells are particularly useful in the treatment of relapsed or refractory cancer in human patients. Also provided are methods of selecting patients who will benefit from prevention or treatment of cancer through the medical uses or methods of treatment of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/305,162, filed Nov. 28, 2018, which is a 371 national stageapplication based on Patent Cooperation Treaty Application serial numberPCT/GB2017/051560, filed May 31, 2017, which claims the benefit ofpriority to United Kingdom Patent Application No. GB 1609600.0, filedJun. 1, 2016. The entirety of each of these applications is herebyincorporated by reference herein for all purposes.

FIELD OF THE INVENTION

The invention relates to medical uses and methods for targeting cancerstem cells, particularly in the prevention or treatment of cancer. Thepresent invention also relates to medical uses and methods for thetreatment of relapsed or refractory cancer in human patients. Theinvention provides methods of selecting patients who will benefit fromprevention or treatment of cancer through the medical uses or methods oftreatment of the invention.

INTRODUCTION

The putative existence of a cancer stem cell has been suggested in manyhuman cancers including leukaemias and solid tumours. The cancer stemcell hypothesis considers that only a small sub-population of tumourcells is responsible for the formation and maintenance of the bulk ofthe tumour. The emergence of this concept can be traced back to the workof Lapidot et al. (1994), who showed evidence that only a smallpercentage of acute myeloid leukaemia cells had the capability toinitiate leukaemia in mice. These cells were shown to express similarcell surface markers (CD34⁺/CD38⁻) to normal haematopoietic stem cellsimplying that a similar hierarchical organisation may occur in tumours.Subsequently, cancer stem cells have been identified in a wide range ofsolid tumours including breast, lung, colon, prostate, ovarian, skin,and pancreas.

Conventional anti-cancer approaches are directed predominantly at bulktumour populations. Such strategies often have limited efficacy becauseof intrinsic or acquired drug resistance and/or resistance to ionizingradiation, so relapse and the emergence of drug resistance are commonfeatures of many cancers. Mechanisms of therapeutic resistance includeincreased recognition and repair of therapy-induced DNA damage, alteredcell cycle checkpoint control, impaired functioning of apoptoticpathways, and reduced drug accumulation as a result of increasedexpression of ABC transporters that efflux drugs. Evidence has emergedthat cancer stem cells have increased resistance to chemo- andradiotherapy and are often enriched for in patients who relapse. Overtcancer stem cell chemo-resistance has been reported in human leukaemias,in malignant melanoma, and in several solid tumours including breast,pancreatic, and colorectal cancers.

Cancer stem cell-specific phenotypes and functions have been shown tocontribute to tumourigenicity, cancer progression, and therapeuticresistance. The persistence of cancer stem cells may also contribute totreatment failure. Therefore, cancer stem cells represent novel andtranslationally relevant targets for cancer therapeutics.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides a ProTide compound, as defined herein, for use intargeting cancer stem cells.

The invention provides the use of a ProTide compound, as defined herein,in the manufacture of a medicament for targeting cancer stem cells.

The invention provides a method of targeting cancer stem cells, themethod comprising providing a population of cancer stem cells with anamount of a ProTide compound, as defined herein, sufficient to targetsuch cancer stem cells.

The targeting of cancer stem cells referred to in the present inventionmay be employed in the prevention or treatment of cancer. In suchembodiments the population of cancer stem cells may be in a cancer orpre-cancerous condition in a patient in need of such targeting, and themethod may comprise administering a therapeutically effective amount ofa ProTide compound, as defined herein, to the patient.

The invention provides a ProTide compound, as defined herein, for use asan anti-cancer stem cell medicament. This use of a ProTide compound, asdefined herein, may also be employed in the prevention or treatment ofcancer.

The invention provides a method of determining whether a patient withcancer or a pre-cancerous condition will benefit from prevention ortreatment of cancer with a compound of the invention, the methodcomprising:

assaying a biological sample representative of cancer or a pre-cancerouscondition in the patient for the presence of cancer stem cells; whereinthe presence of cancer stem cells in the biological sample indicatesthat the patient will benefit from treatment with a compound of theinvention.

The invention provides a method of determining a suitable treatmentregimen for a patient with cancer or a pre-cancerous condition, themethod comprising:

assaying a biological sample representative of cancer or a pre-cancerouscondition in the patient for the presence of cancer stem cells; whereinthe presence of cancer stem cells in the biological sample indicatesthat a suitable treatment regimen will comprise treatment of the patientwith a compound of the invention.

The invention provides a ProTide compound, as defined herein, for use inthe prevention or treatment of cancer in a patient selected for suchtreatment by a method comprising:

assaying a biological sample representative of cancer or a pre-cancerouscondition in the patient for the presence of cancer stem cells; whereinthe presence of cancer stem cells in the biological sample indicatesthat the patient is suitable for treatment with a compound of theinvention.

The methods set out above may further comprise a step of preventing ortreating the cancer or pre-cancerous condition using a compound of theinvention.

In suitable embodiments of the methods of the invention the cancer isrelapsed or refractory cancer. A ProTide compound, as defined herein,may be used for the treatment of such relapsed or refractory cancer.

The invention provides a ProTide compound, as defined herein, for use intreatment of refractory cancer in a subject. The invention provides aProTide compound, as defined herein, for use in treatment of relapsedcancer in a subject. The subject may be a human patient.

The invention provides the use of a ProTide compound, as defined herein,in the manufacture of a medicament for the treatment of relapsed orrefractory cancer in a human patient.

The invention provides a method of treating relapsed or refractorycancer in a subject, the method comprising providing a therapeuticallyeffective amount of a ProTide compound, as defined herein, to a subjectin need of such treatment.

The invention provides a ProTide compound, as defined herein, for use inthe treatment of cancer, wherein the ProTide compound is for use at doseof between approximately 500 mg/m² and 1000 mg/m² per week in at leastone initial cycle of treatment, and then for use at a lower weekly dosein at least one further cycle of treatment. The cancer may be a relapsedor refractory cancer.

The invention also provides a ProTide selected from: CPF447, CPF727,CPF544, CPF680, CPF545 and CPF682, and CPF792, or a pharmaceuticallyacceptable salt thereof. The invention also provides a pharmaceuticalformulation comprising a ProTide selected from: CPF447, CPF727, CPF544,CPF680, CPF545 and CPF682, and CPF792, or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable excipient. The ProTidemay be selected from CPF680, CPF544, CPF682 and CPF792, or apharmaceutically acceptable salt thereof. The ProTide may be selectedfrom: CPF447, CPF727, CPF544, CPF680, CPF545 and CPF682, and CPF792. TheProTide may be selected from CPF680, CPF544, CPF682 and CPF792. Each ofthese ProTides may be used in targeting of cancer stem cells, and thusthese ProTides represent suitable ProTides for use in accordance withthe various aspects and embodiments of the invention set out above.

DETAILED DESCRIPTION OF THE INVENTION

Various aspects of the invention are based upon the finding that aProTide compound, as defined herein, is able to preferentially reducecancer stem cell numbers. This finding is surprising in that cancer stemcells are known to be resistant to many chemotherapeutic agents, andthere has previously been no suggestion that the ProTide compounds thatare the subject of the present invention were able to target cancer stemcells. Thus the finding that a ProTide compound, as defined herein, isable to target cancer stem cells and thus reduce their numbers, afinding which the inventors have confirmed is applicable across a broadrange of cancers, represents a surprising breakthrough that enables arange of new therapeutic applications of a compound of the invention.

The biological activities exerted by the compounds of the invention,which are described elsewhere in the present specification and whichhave not previously been reported, indicate that these compounds areable to provide treatment that is likely to be effective in patientswith relapsed or refractory cancers. Treatment of this sort, using thecompounds of the invention, may bring about a reduction in tumour sizeand/or a reduction in clinically relevant biomarkers, either of whichmay be associated with more favourable prognosis. Furthermore, treatmentwith a ProTide compound, as defined herein, may help to maintain areduction in the size of tumours in patients with relapsed or refractorycancer. Accordingly, treatment using a ProTide compound, as definedherein, may achieve a high, durable Disease Control Rate (DCR) inpatients with relapsed or refractory cancers.

Without wishing to be bound by any hypothesis, the inventors believethat the ability of the compounds of the invention to target cancer stemcells contributes to the therapeutic utility of these compounds in thetreatment of relapsed or refractory cancer.

Except for where the context requires otherwise, references within thisdisclosure to a “use” of a ProTide compound, as defined herein, inaccordance with the invention may be taken as applying to any of themedical uses of compounds of the invention described herein. Similarly,references to “methods” of the invention using a ProTide compound, asdefined herein, should be taken as applying to any of the methods of theinvention herein described.

The ability of a ProTide compound, as defined herein, to target cancerstem cells enables new therapies directed against those cancer cellsthat are considered most difficult to treat, and that are considered toplay a major role in the resistance that limits effectiveness of manyexisting cancer therapies. This ability also provides a way of targetingcells that are believed to be associated with the development,progression, recurrence, and propagation of cancers. Accordingly, itwill be recognised that this anti-cancer stem cell activity of a ProTidecompound, as defined herein, yields benefits in contexts in which newand effective therapies have long been sought.

The present invention is, to a great extent, based upon the inventors'findings regarding the biological activities exerted by a group ofProTide compounds. As described further below, the biological activitiesof these ProTide compounds clearly indicate that they will havetherapeutic activity in the various clinical contexts considered herein.The uses of ProTide compounds in accordance with the various aspects ofthe present invention are of particular utility in the prevention ortreatment of cancer, and even of relapsed or refractory cancer.

The inventors have found that the ‘ProTide’ compoundgemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate (also referred to asNUC-1031, or by the trade name Acelarin), which is a ProTide derivativeof the chemotherapeutic agent gemcitabine, is able to provide effectivetreatment of cancer, in clinical studies, including effective treatmentof relapsed or refractory cancer. The effectiveness of NUC-1031 intreatment of cancer in this manner appears to derive from the ability ofthis compound to target cancer stem cells in the treatment of cancer.The effective treatment of cancer provided by the ProTide NUC-1031 mayresult in reduced number of cancer stem cells, slower diseaseprogression, reduction in tumour size and a reduction in cancerbiomarker levels, such as carcinoembryonic antigen (CEA), cancer antigen(CA) 19.9, CA 125 and inhibin B. Details of the clinical studiesdemonstrating the therapeutic utility of NUC-1031 are set out inComparative Example 2 of the present application.

This therapeutic utility of NUC-1031 arises from the biological activityof this compound. NUC-1031 is cytotoxic for cancer stem cells in vitro.Furthermore, this effect is found to be selective—which is to say thatwhile NUC-1031 is cytotoxic for both cancer stem cells and non-stemcancer cells, it exerts this effect to a greater extent on cancer stemcells. Accordingly, in mixed populations of cancer stem cells andnon-stem cancer cells, treatment with NUC-1031 reduces the proportion ofcancer stem cells present below the levels found prior to treatment.Comparison of the effects of NUC-1031 and the parent compoundgemcitabine on mixed populations of cancer stem cells and non-stemcancer cells indicates that the selectivity demonstrated by NUC-1031 isstatistically significantly greater than the targeting by the parentcompound. The inventors believe that these in vitro activities ofNUC-1031 equate to treatment of cancer observed on provision of thiscompound in vivo.

As set out elsewhere in the specification, the definition of ProTidecompounds suitable for use in accordance with the present inventionexcludes NUC-1031, and may also be taken as excluding protide compoundsbased upon the anti-cancer agents cordycepin and 8-chloroadenosine.Thus, a protide compound in accordance with the present invention may bea ProTide compound other than one selected from the group consisting of:NUC-1031; a ProTide derived from cordycepin; and a ProTide derived from8-chloroadenosine.

As demonstrated in the results set out elsewhere in the presentspecification, the inventors have shown that ProTide compounds otherthan NUC-1031 are able to target cancer stem cells and thereby reducethe proportion of such cells within populations of cells treated withthe ProTide. Examples of ProTide compounds shown to be suitable for suchuses include those selected from the group consisting of: ProTidecompounds derived from FUDR, in particular ProTide compounds selectedfrom the group consisting of: CPF-373 (whether Isomer A, or Isomer B),CPF-381, CPF-581, and CPF-585; ProTide compounds derived fromthioguanosine, in particular the ProTide compounds CPF-775 or CPF-782;ProTide compounds derived from cladribine, in particular the ProTidecompounds CPF-793 and CPF-791; ProTide compounds derived fromthioinosine, in particular the ProTide compounds CPF-761 and CPF-762;ProTide compounds derived from fludarabine, in particular the ProTidecompounds CPF-544 and CPF-682; and ProTide compounds derived fromclofarabine, in particular ProTide compounds selected from the groupconsisting of: CPF-720, CPF-727, and CPF-448.

Furthermore, the inventors have shown that certain ProTide compoundsother than NUC-1031 are able to target cancer stem cells moreeffectively than the parent anti-cancer compounds from which they arederived. Such ProTide compounds represent very promising agents fortherapeutic uses described herein, and include those selected from thegroup consisting of: ProTide compounds derived from FUDR, in particularProTide compounds selected from the group consisting of: CPF-373(whether Isomer A, or Isomer B), CPF-381, and CPF-581; ProTide compoundsderived from thioguanosine, in particular the ProTide compounds CPF-775or CPF-782; ProTide compounds derived from cladribine, in particular theProTide compound CPF-793; and ProTide compounds derived fromthioinosine, in particular the ProTide compound CPF-761.

The inventors have also shown that certain ProTide compounds other thanNUC-1031 are able to not only target cancer stem cells more effectivelythan the parent anti-cancer compounds from which they are derived, butalso demonstrate greater anti-cancer potency than the parent compoundsfrom which they are derived. This combination of activities is shown byNUC-1031 in vitro, and the ability of these ProTide compounds, asdefined herein, to achieve the same profile of activities in vitro makesthese compounds particularly promising agents to achieve the sameremarkable clinical effects noted in respect of NUC-1031. Such ProTidecompounds include those selected from the group consisting of: ProTidecompounds derived from FUDR, in particular ProTide compounds selectedfrom the group consisting of: CPF-373 (whether Isomer A, or Isomer B),CPF-381, and CPF-581.

As described herein, the inventors have found that ProTides, includingthose selected from the group consisting of: CPF-373 (Iso A), CPF-373(Iso B), CPF-381, CPF-581, CPF-782, CPF-775, CPF-544, CPF-682, CPF-793,CPF-448, CPF-585, CPF-791, CPF-720, CPF-727, CPF-761, and CPF-762,selectively target for cancer stem cells, as opposed to non-stem cancercells.

ProTides, including those selected from the group consisting of: CPF-373(Iso A), CPF-373 (Iso B), CPF-381, CPF-581, CPF-782, CPF-775, CPF-544,CPF-682, CPF-793 and CPF-448, show high selectivity for cancer stemcells.

Finally, ProTides, including those selected from the group consisting ofCPF-373 (Iso A), CPF-373 (Iso B), CPF-381, CPF-581 and CPF-782, showhigh selectivity for cancer stem cells, and surprisingly, also greaterpotency as compared to the respective parent compounds from which theyare derived from.

These findings, which have not previously been reported, indicate thatProTides other than NUC-1031 will be able to exert the same utility inthe targeting of cancer stem cells for the treatment or prevention ofcancer. Thus these other ProTide compounds may provide the sameadvantages, through targeting cancer stem cells in the treatment ofcancer, that have been shown to be effective in respect of NUC-1031.

Definitions ProTide Compounds

The term ‘ProTide’ is readily understood in the art to mean an aryloxyphosphoramidate derivative of a nucleoside or nucleoside analogue. Thus,the ProTide may be a compound having a structure according to formula(I):

(I) wherein

R¹ is aryl;

R² is C₁-C₂₄-alkyl, C₃-C₂₄-alkenyl, C₃-C₂₄-alkynyl, C₀-C₄alkylene-C₃-C₆-cycloalkyl or C₀-C₄-alkylene-aryl;

R³ and R⁴ are each independently selected from H, C₁-C₆-alkyl andC₁-C₃-alkylene-R⁶; or wherein R³ and R⁴ together with the atom to whichthey are attached form a 3- to 6-membered cycloalkyl or heterocycloalkylgroup;

R⁵ is a nucleoside or nucleoside analogue;

R⁶ is independently selected from aryl (e.g. phenyl), imidazole, indole,SR^(a), OR^(a), CO₂R_(a), CO₂NR^(a)R^(a), NR^(a)R^(b) and NH(═NH)NH₂;

wherein any aryl group is either phenyl or naphthyl and wherein anyphenyl or naphthyl group is optionally substituted with from 1 to 4substituents selected from: halo, nitro, cyano, NR^(a)R^(a),NR^(a)S(O)₂R^(a), NR^(a)C(O)R^(a), NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a),OR^(a); SR^(a), SOR^(a), SO₃R^(a), SO₂R^(a), SO₂NR^(a)R^(a), CO₂R^(a)C(O)R^(a), CONR^(a)R^(a), CR^(a)R^(a)NR^(a)R^(a), C₁-C₄-alkyl,C₂-C₄-alkenyl, C₂-C₄-alkynyl and C₁-C₄-haloalkyl;

wherein R^(a) is independently at each occurrence selected from: H andC₁-C₄-alkyl; and R^(b) is independently at each occurrence selectedfrom: H, and C₁-C₄-alkyl and C(O)—C₁-C₄-alkyl. NUC-1031(gemcitabine-[phenyl-benzoxy-L-alaninyl)]-phosphate or, to give it itsfull chemical name: 2′-deoxy-2′,2′-difluoro-D-cytidine-5′-O-[phenyl(benzoxy-L-alaninyl)] phosphate) is however explicitly excluded from thescope of this application.

Compounds of formula (II) are also explicitly excluded from the scope ofthis application:

wherein:W₁ and W₂ are each independently selected from the group consisting of—P(═O)(U)(V) and H, with the proviso that at least one of W₁ and W₂ is—P(═O)(U)(V),where U and V, independently for each of W₁ and W₂, are selected fromthe group consisting of:(a) U is —OAr in combination with V is —NR_(D)—CR_(A)R_(B)—C(═O)OR_(C),where Ar is selected from the group consisting of C₆₋₃₀aryl and₅₋₃₀heteroaryl, each of which is optionally substituted;each of R_(A) and R_(B) is independently selected from H, and the groupconsisting of C₁₋₂₀alkyl, C₆₋₃₀arylC₁₋₆alkyl, C₂₋₂₀alkenyl, C₁₋₂₀alkoxy,C₁₋₂₀alkoxyC₁₋₂₀alkyl, C₁₋₂₀alkoxyC₆₋₃₀aryl, C₂₋₂₀alkynyl,C₃₋₂₀cycloalkylC₆₋₃₀aryl, C₆₋₃₀aryloxy and ₅₋₂₀heterocyclyl, any ofwhich is optionally substituted;R_(C) is selected from H, and the group consisting of C₁₋₂₀alkyl,C₆₋₃₀arylC₁₋₂₀alkyl, C₂₋₂₀alkenyl, C₁₋₂₀alkoxyC₁₋₂₀alkyl,C₁₋₂₀alkoxyC₆₋₃₀aryl, C₂₋₂₀alkynyl, C₃₋₂₀cycloalkylC₆₋₃₀aryl, and₅₋₂₀heterocyclyl, any of which is optionally substituted;R_(D) is selected from H, and the group consisting of C₁₋₂₀alkyl,C₆₋₃₀arylC₁₋₂₀alkyl, C₂₋₂₀alkenyl, C₁₋₂₀alkoxy, C₁₋₂₀alkoxyC₁₋₂₀alkyl,C₁₋₂₀alkoxyC₆₋₃₀aryl, C₂₋₂₀alkynyl, C₃₋₂₀cycloalkylC₆₋₃₀aryl,C₆₋₃₀aryloxy and ₅₋₂₀heterocyclyl, any of which is optionallysubstituted;and(b) each of U and V is selected independently from —NR_(E)R_(F),where R_(E) is selected from the group consisting of H and C₁₋₆alkyl andR_(F) is —CR_(G)R_(H)CO₂R_(I), where R_(G) and R_(H) are selectedindependently from the group consisting of the side chains, including H,of naturally occurring alpha amino acids and R_(I) is selected from H,and the group consisting of C₁₋₂₀alkyl, C₆₋₃₀arylC₁₋₂₀alkyl-,C₂₋₂₀alkenyl, C₁₋₂₀alkoxyC₁₋₂₀alkyl, C₁₋₂₀alkoxyC₆₋₃₀aryl, C₂₋₂₀alkynyl,C₃₋₂₀cycloalkylC₆₋₃₀aryl, and ₅₋₂₀heterocyclyl, any of which isoptionally substituted; or R_(E) and R_(F) together with the N atom towhich they are attached form a ring moiety comprising 5 to 8 ring atoms;Q is selected from the group consisting of O, S and CR_(J)R_(K), whereR_(J) and R_(K) are independently selected from H, F and C₁₋₆alkyl;each of X and Z is independently selected from the group consisting ofH, OH, F, Cl, Br, I, C₁₋₆alkyl, —NR_(L)R_(M) where each of R_(L) andR_(M) is independently selected from H and C₁₋₆alkyl, and —SR_(N) whereR_(N) is selected from the group consisting of H and C₁₋₆alkyl; andY is selected from the group consisting of H, OH, F, Cl, Br, I,—OC₁₋₆alkyl, C₁₋₆alkyl, C₂₋₈alkynyl, —NR_(O)R_(P) where each of R_(O)and R_(P) is independently selected from H and C₁₋₆alkyl, and —SR_(Q)where R_(Q) is selected from the group consisting of H and C₁₋₆alkyl,or a pharmaceutically acceptable salt, ester, salt of an ester, solvateor prodrug of the compound of formula (II).The nucleoside or nucleoside analogue may have the structure:

wherein:

Q is independently selected from O, NR^(a) and CH₂;

R⁷ is independently selected from: OR^(a), SR^(a), NR^(a)R^(b); halo(e.g. F), cyano, C₁-C₄-alkyl, C₂-C₄-alkenyl and C₂-C₄-alkynyl;

R⁸ and R⁹ together with the nitrogen to which they are attached form asubstituted pyrimidine or a substituted purine; wherein the purine orpyrimidine is substituted with from 1 to 5 groups selected from: OR^(a),SR^(a), NR^(a)R^(b); halo, cyano, C₁-C₄-alkyl, C₂-C₄-alkenyl andC₂-C₄-alkynyl.

n is typically an integer from 0 to 4. n may be an integer from 1 to 3.As will be readily appreciated by the skilled person, where a pyrimidineor purine is substituted with an OH group attached to a carbon atomneighbouring one of the nitrogen atoms in the pyrimidine or purine core,the pyrimidine or purine will typically exist primarily in thetautomeric form, i.e. one in which there is no double bond between thenitrogen and the neighbouring carbons but in which there is a doublebond between the neighbouring carbon and the oxygen of the OH group.Said nitrogen may itself be substituted, e.g. with a C₁-C₄-alkyl group.The ProTide may be a compound having a structure according to formula(III):

(III) wherein

R¹, R², R³ and R⁴ are as described above for compounds of formula (I).NUC-1031 is excluded from the scope of this application and thus, forthe absence of doubt, where the ProTide is a compound of formula (III),it cannot be the case that R¹ is unsubstituted phenyl, R² isunsubstituted benzyl, R³ is Me and R⁴ is H. ProTides of formula (III)are derivatives of gemcitabine.

The ProTide may be a compound having a structure according to formula(IV):

R¹, R², R³ and R⁴ are as described above for compounds of formula (I).ProTides of formula (IV) are derivatives of FUDR.

The ProTide may be a compound having a structure according to formula(V):

R¹, R², R³ and R⁴ are as described above for compounds of formula (I).ProTides of formula (V) are derivatives of clofarabine.

The ProTide may be a compound having a structure according to formula(VI):

R¹, R², R³ and R⁴ are as described above for compounds of formula (I).ProTides of formula (VI) are derivatives of fludarabine.

The ProTide may be a compound having a structure according to formula(VII):

wherein

R¹, R², R³ and R⁴ are as described above for compounds of formula (I).ProTides of formula (VII) are derivatives of cladribine.

The following statements apply to ProTides of any of formulae (I) and(III) to (VII). These statements are independent and interchangeable. Inother words, any of the features described in any one of the followingstatements may (where chemically allowable) be combined with thefeatures described in one or more other statements below. In particular,where a compound is exemplified or illustrated in this specification,any two or more of the statements below which describe a feature of thatcompound, expressed at any level of generality, may be combined so as torepresent subject matter which is contemplated as forming part of thedisclosure of this invention in this specification.

It may be that R¹ is substituted or unsubstituted phenyl. It may be thatR¹ is substituted or unsubstituted naphthyl (e.g. 1-naphthyl).Preferably, R¹ is unsubstituted phenyl or unsubstituted naphthyl (e.g.1-naphthyl). Thus, R¹ may be unsubstituted phenyl. Alternatively, R¹ maybe or unsubstituted naphthyl (e.g. 1-naphthyl).

R² is preferably selected such that it comprises five or more carbonatoms. R² may therefore be selected such that it includes six or morecarbon atoms. R² is preferably selected such that it comprises onlycarbon and hydrogen atoms. R² may be selected from C₅-C₇-cycloalkyl,C₅-C₈-alkyl and benzyl, optionally wherein said groups areunsubstituted.

It may be that R⁴ is H. It may be that R³ is selected from C₁-C₆-alkyland C₁-C₃-alkylene-R⁶. It may be that R³ is C₁-C₄-alkyl. It may be thatR³ is methyl.

Q is preferably O.Exemplary ProTides of formula (III) include the compounds described inWO 2005/012327, incorporated herein by reference.

Exemplary ProTides of formula (IV) include the compounds described in WO2012/117246, incorporated herein by reference. Exemplary ProTides offormula (IV) include:

Exemplary ProTides of formulae (V), (VI) and (VII) include the compoundsdescribed in WO2006/100439. Exemplary ProTides of formula (V) include:

Exemplary ProTides of formula (VI) include:

Exemplary ProTides of formula (VII) include:

Further exemplary ProTide compounds described herein include:

“Targeting of Cancer Stem Cells”

The present invention provides the first indication that compounds ofthe invention can be used for targeting cancer stem cells. The abilityof compounds of the invention to target cancer stem cells is illustratedin the studies investigating the proportion of CD34⁺/CD38⁻/CD123⁺ cellssurviving treatment with different concentrations of ProTide compounds,which are set out in the Examples disclosed elsewhere in thisspecification.

It can be seen from the results reported in the Examples that when aProTide compound, as defined herein (ProTide compounds derived fromFUDR, in particular ProTide compounds selected from the group consistingof: CPF-373 (whether Isomer A, or Isomer B), CPF-381, CPF-581, andCPF-585; ProTide compounds derived from thioguanosine, in particular theProTide compounds CPF-775 or CPF-782; ProTide compounds derived fromcladribine, in particular the ProTide compounds CPF-793 and CPF-791;ProTide compounds derived from thioinosine, in particular the ProTidecompounds CPF-761 and CPF-762; ProTide compounds derived fromfludarabine, in particular the ProTide compounds CPF-544 and CPF-682;and ProTide compounds derived from clofarabine, in particular theProTide compounds CPF-720, CPF-727, and CPF-448) is provided topopulations of cancer cells containing cancer stem cells it targets thecancer stem cells present, leading to a reduction in the total number ofcancer cells and in the proportion of total cancer cells exhibitingphenotypic markers of cancer stem cells.

Certain ProTide compounds exhibit an ability to target cancer stem cellsthat is significantly increased as compared to the parent compound fromwhich they are derived. These include ProTides selected from the groupconsisting of: ProTide compounds derived from FUDR, in particularProTide compounds selected from the group consisting of: CPF-373(whether Isomer A, or Isomer B), CPF-381, and CPF-581; ProTide compoundsderived from thioguanosine, in particular the ProTide compounds CPF-775or CPF-782; ProTide compounds derived from cladribine, in particular theProTide compound CPF-793; and ProTide compounds derived fromthioinosine, in particular the ProTide compound CPF-761.

Without wishing to be bound by any hypothesis, the inventors believethat the reduction in cancer stem cell numbers arises as a result oftargeted killing of the cancer stem cells among the cancer cellpopulation. That is to say, that compounds of the invention appear tokill cancer stem cells preferentially as compared to killing of non-stemcancer cells, thereby causing the death of cancer stem cells, and areduction of the proportion of cancer stem cells among the total cancercell population.

While the inventors believe that compounds of the inventionpreferentially kills cancer stem cells as compared to non-stem cancercells, other mechanisms may also contributed to the reduction in theproportion of cancer stem cells caused by a compound of the invention'stargeting of these cells.

Merely by way of example, treatment with a ProTide compound, as definedherein, may cause an increase in cancer stem cell differentiation,thereby reducing cancer stem cell numbers and also the proportion oftotal cancer cells represented by cancer stem cells. Alternatively, aProTide compound, as defined herein, may cause cancer stem cells to losetheir stem cell phenotype, for example losing their ability toself-renew, thereby reducing cancer stem cell numbers.

References to targeting of cancer stem cells in the present disclosureshould be interpreted accordingly. For the purposes of the presentdisclosure, “targeting” of cancer stem cells may be taken asencompassing any mechanism by which a ProTide compound, as definedherein, reduces the proportion of cancer stem cells present in apopulation of cells, whether in vitro or in vivo. In particulartargeting of cancer stem cells may be taken as encompassing preferentialkilling of cancer stem cells as compared to other cell types,particularly as compared to non-stem cancer cells.

“Cancer Stem Cells”

Cancer stem cells, which are sometimes otherwise referred to as “tumourinitiating cells”, are well known to those skilled in the art. As usedherein, the term “cancer stem cell” is to be interpreted in accordancewith its widely accepted meaning, which is a cell that possesses thecapacity to self-renew through asymmetric division, to initiate tumourformation, and to give rise to more mature non-stem cell cancer progenyby differentiation.

Cancer stem cells play a major role in the development, progression,recurrence and propagation of cancers. Accordingly, the finding thatcompounds of the invention are able to target cancer stem cells, andthereby reduce their numbers, offers therapeutic possibilities inpreventing or treating these activities.

As discussed in more detail elsewhere in the specification, cancer stemcells are found in pre-cancerous conditions, where their presence isbelieved to contribute to the development of such conditions intocancers. Accordingly the methods of treatment and medical uses of theinvention, in which a ProTide compound, as defined herein, is used totarget cancer stem cells, may be used to reduce cancer stem cell numbersin pre-cancerous conditions (such as myelodyplastic syndrome, or otherconditions considered elsewhere in the specification), and thus toprevent progression of such pre-cancerous conditions into cancer.

As referred to above, asymmetric cell division of cancer stem cellsgives rise to differentiated non-stem cancer cells. Thus cancer stemcells are responsible for the formation and maintenance of the bulk ofthe tumour.

The accumulation of such non-stem cancer cells plays a major role in theprogression of cancers. Targeting of cancer stem cells by a ProTidecompound, as defined herein, is able to reduce cancer stem cell numbers,which in turn reduces the number of non-stem cancer cell progeny. Thusmethods of treatment and medical uses of a ProTide compound, as definedherein, in accordance with the present invention are of benefit intreating cancer by preventing cancer progression. Such embodiments aredescribed in more details elsewhere in the present specification.

Cancer stem cells are also able to act as a reservoir of cancer cellsthat they may cause the recurrence of cancer after remission. Even inthe event that the majority of a patient's cancer cells have beenremoved (for example by surgery, radiotherapy, or chemotherapy, eitheralone or in combination), so that no observable signs of a cancerremain, the continued presence of cancer stem cells may nucleate therecurrence of the cancer over time. Targeting of cancer stem cells by aProTide compound, as defined herein, provides a new mode by which cancerstem cell numbers may be reduced and cancer stem cells killed.Accordingly, and as discussed in more detail elsewhere in thespecification, in suitable embodiments the present invention providesmethods and medical uses in which a ProTide compound, as defined herein,prevents or delays recurrence of cancer.

Furthermore, movement of cancer stem cells from the site of a cancer toanother location within the body can contribute to propagation ofcancer, for example by giving rise to metastases. Consequently, theability of a ProTide compound, as defined herein, to target cancer stemcells therefore provides new methods of treatment and medical uses inpreventing or treating cancer propagation.

In addition to their biological activities, cancer stem cells may beidentified by their expression of certain characteristic cell surfacemarkers. Cancer stem cells identified in haematological malignancies aretypically CD34⁺, while in solid tumours, CD44⁺, CD133⁺ and CD90⁺ havebeen identified as cancer stem cell markers. The following tablesummarises examples of known cancer stem cell surface phenotypes. It isexpected that each of these forms of cancer stem cell can be targetedusing a ProTide compound, as defined herein, in accordance with theinvention, and so methods or uses employing such ProTide compounds maybe used in the prevention or treatment of cancers associated with cancerstem cells expressing any of these sets of markers.

Reported cell surface markers for Tumour type cancer stem cells SolidTumours Breast CD44⁺/CD24⁻/^(low)/Lineage⁻/ESA⁺ CNS CD133⁺ Colon CD133⁺Colon ESA^(high)/CD44⁺/Lineage⁻/(CD166⁺) Ewing's CD133⁺ Head and NeckCD44⁺/Lineage⁻ Melanoma ABCB5⁺ Liver CD90⁺/CD45⁻/(CD44⁺)Cholangiocarinoma CD44⁺/GLI1⁺ (Glioma-associated oncogene homolog-1)Ovarian CD44⁺/CD117⁺ Pancreas CD44⁺/CD24⁺/ESA⁺ Pancreas CD133⁺Non-small-cell lung cancer CD44⁺/Ber-EP4⁺ Bladder cancer CD44⁺/ALDH1A1⁺Haematological tumours Acute myeloid leukaemia Lin⁻/CD34⁺/CD38⁻/CD123⁺B-Acute lymphoblastic leukaemia CD34⁺/CD10⁻ or CD34⁺/CD19⁻ B-Acutelymphoblastic leukaemia CD34⁺/CD38⁻/CD19⁺ Multiple myeloma CD34⁻/CD138⁻T-Acute lymphoblastic leukaemia CD34⁺/CD4⁻ or CD34⁺/CD7⁻

The data presented in the Examples demonstrate that ProTide compounds,as defined herein, are able to target cancer stem cells of leukaemicstem cell lines, specifically cancer stem cells present in the acutemyeloid leukaemia cell line KG1a. This cell line manifests a minor stemcell-like compartment with a distinct immunophenotype(Lin⁻/CD34⁺/CD38⁻/CD123⁺), which is targeted by the ProTides definedherein. Accordingly, methods of treatment or medical uses of a ProTidecompound, as defined herein, in accordance with the present inventionmay be used to prevent or treat leukaemia or other cancers associatedwith cancer stem cells expressing these characteristic markers.

The present invention also provides methods and medical uses in whichpatients are selected for prevention or treatment of cancer, utilising acompound of the invention, on the basis of the identification of thepresence of cancer stem cells in a biological sample representative ofthe patient's cancer or pre-cancerous condition. The markers set outabove provide suitable examples that can be used to identify thepresence of cancer stem cells in accordance with such embodiments of theinvention. Suitable techniques by which expression of these markers maybe investigated in a biological sample are considered further elsewherein this specification.

“Prevention or Treatment of Cancer”

The invention provides medical uses and methods of treatment in which aProTide compound, as defined herein, is used for the prevention ortreatment of cancer. In the context of the present invention,“prevention” of cancer is to be considered as relating to prophylacticapplications of a ProTide compound, as defined herein, used before thedevelopment of cancer, and with an aim of stopping cancer fromdeveloping. On the other hand “treatment” of cancer is taken asconcerning the use of a ProTide compound, as defined herein, aftercancer has occurred, with a view to ameliorating cancer by slowing orstopping cancer cell proliferation and tumour growth. Advantageouslytreatment of cancer may cause partial or total reduction in cancer cellnumbers and tumour size. Effective treatment of cancer may bring aboutdisease that either “stabilizes” or “responds” in accordance with theRECIST (Response Evaluation Criteria In Solid Tumours) rules.

As described in more detail below, prevention of cancer in accordancewith the present invention may be of particular benefit in patients whohave a pre-cancerous condition that increases their likelihood ofdeveloping cancer.

“Prevention of Cancer”

Prevention of cancer in accordance with the present invention may beeffected by treatment of a pre-cancerous condition using a ProTidecompound, as defined herein, in accordance with the various aspects orembodiments of the invention described herein.

In particular, prevention of cancer, in the context of the presentinvention, may be achieved by the methods or medical uses of theinvention in which a ProTide compound, as defined herein, is provided toa patient with a pre-cancerous condition. Methods of treatment ormedical uses in accordance with this embodiment may prevent developmentof the treated pre-cancerous condition into cancer, thereby providingeffective prevention of cancer.

References to prevention of cancer in the context of the presentinvention may also encompass other prophylactic applications of acompound of the invention. For example, the ability of a ProTidecompound, as defined herein, to target cancer stem cells and therebyprevent the development of cancer, and/or prevent the progression ofcancer, and/or prevent the recurrence of cancer, and/or prevent thepropagation of cancer.

“Pre-Cancerous Conditions”

Cancer is frequently preceded by the development of a pre-cancerouscondition, which is not itself cancerous, but is associated with anincreased risk of cancer. Accumulation of genetic or epigenetic changesmay cause previously normal cells to develop a cancer stem cellphenotype. Accordingly, cancer stem cells may also be present in suchpre-cancerous conditions, as well as in cancerous conditions.

It is believed that the presence of cancer stem cells in pre-cancerousconditions contributes to the development of these conditions intocancer. The methods and medical uses of the invention may be employed totarget cancer stem cells present in pre-cancerous conditions, andthereby treat such conditions. It will be appreciated that the new andunexpected finding that compounds of the invention target cancer stemcells means that treatment of pre-cancerous conditions with suchcompounds may be used to prevent the treated conditions developing intocancer. This represents a way in which a ProTide compound, as definedherein, can be used medically in the prevention of cancer, as consideredelsewhere in this specification.

Examples of pre-cancerous conditions that may be treated in accordancewith the present invention include, but are not limited to, thoseselected from the group consisting of: actinic keratosis, Barrett'soesophagus, atrophic gastritis, dyskeratosis congenital, Sideropenicdysphagia, Lichen planus, oral submucous fibrosis, solar elastosis,cervical dysplasia, leukoplakia, erythroplakia, monoclonal gammopathy ofunknown significance (MGUS), monoclonal B-cell lymphocytosis (MBL),myelodysplastic syndromes, as well as pre-cancerous conditions of thestomach such as atrophic gastritis, gastric ulcer, pernicious anaemia,gastric stumps, gastric polyps, and Ménétrier's disease. Among thelisted pre-cancerous conditions of the stomach, atrophic gastritis,pernicious anaemia, gastric stumps, and certain types of gastric polypmay have particularly heightened risk of developing into cancers.

Pre-cancerous conditions often take the form of lesions comprisingdysplastic or hyperplastic cells. Accordingly, the presence of dysplasiaor hyperplasia, as an alternative or addition to the presence of cellswith expressed markers or phenotypes characteristic of cancer stemcells, may be used in the identification of pre-cancerous conditions.

The severity of dysplasia can vary between different pre-cancerousconditions, or with the development of a single pre-cancerous conditionover time. Generally, the more advanced dysplasia associated with apre-cancerous condition is, the more likely it is that the pre-cancerouscondition will to develop into cancer. Dysplasia is typically classifiedas mild, moderate or severe. Severe dysplasia usually develops intocancer if left untreated. Suitably, methods of treatment or medical usesemploying a ProTide compound, as defined herein, may therefore be usedto treat a patient with a pre-cancerous condition associated with severedysplasia.

In a suitable embodiment of the invention a ProTide compound, as definedherein, is used to treat a patient with severe cervical dysplasia.Severe cervical dysplasia may be diagnosed by means of a smear test. Inanother embodiment of the invention a ProTide compound, as definedherein, is used to treat severe oesophageal dysplasia (“Barrett'soesophagus”). Severe oesophageal dysplasia may be diagnosed following atissue biopsy.

It has recently been reported that pre-malignancies can also beidentified by detecting somatic mutations in cells in individuals notknown to have cancer. In particular, it has been reported thatage-related clonal haematopoiesis is a common pre-malignant conditionthat is associated with increased overall mortality and increased riskof cardiometabolic disease. The majority of mutations detected in bloodcells occurred in three genes: DNMT3A, TET2, and ASXL1. Accordingly,patients that will benefit from the use of a ProTide compound, asdefined herein, to target cancer stem cells, and thereby treat apre-cancerous condition, may be identified by assaying a samplecomprising blood cells for the presence of genetic mutations indicativeof a pre-cancerous condition in at least one of: DNMT3A and/or TET2and/or ASXL1.

Pre-cancerous conditions that may benefit from treatment with a ProTidecompound, as defined herein, in accordance with the invention to targetcancer stem cells may also be identified by determination of thepresence of cancer stem cells with reference to any of the techniquesbased upon expression of markers characteristic of cancer stem cells, orcancer stem cell phenotypes, discussed elsewhere in the specification.

“Treatment of Cancer” The skilled person will appreciate that there aremany measurements by which “treatment” of cancer may be assessed. Merelyby way of example, any reduction or prevention of cancer development,cancer progression, cancer recurrence, or cancer propagation may beconsidered to indicate effective treatment of cancer.

In certain embodiments, a ProTide compound, as defined herein, may beused: to reduce the proportion of cancer stem cells in a population ofcancer cells; and/or to inhibit tumour growth; and/or to reducetumourigenicity; and/or to prevent or treat a primary cancer; and/or toprevent or treat a relapsed cancer; and/or to prevent or treat ametastatic or secondary cancer; and/or to treat, prevent or inhibitmetastasis or recurrence; and/or to treat or prevent refractory cancer.

The ability of cancer treatment using a ProTide compound, as definedherein, to bring about a reduction in tumour size, and also to maintainthe reduction in tumour size during/after the period in which thetreatment is administered represents a particularly relevant indicationof effective cancer treatment. As set out in the Examples, thetreatments or medical uses of the invention have proven surprisinglyeffective in this respect, even in models using cells representative ofrelapsed or refractory cancers that have previously been resistant totreatment with other therapies.

The data presented in the Examples illustrate that treatment with aProTide compound, as defined herein, reduces the proportion of cancerstem cells in a population of cancer cells. Characteristic biologicalactivities or cell surface markers by which cancer stem cells may beidentified are described elsewhere in the specification. In a suitableembodiment, treatment of cancer in accordance with the present inventionmay give rise to a reduction in the proportion of cancer stem cellspresent in a patient's cancer of at least 10%, at least 20%, at least30%, or at least 40%. In suitable embodiments treatment of cancer inaccordance with the invention may give rise to a reduction in theproportion of cancer stem cells present in a patient's cancer of atleast 50%, at least 60%, at least 70%, or at least 80%. Treatment ofcancer in accordance with the invention may give rise to a reduction inthe proportion of cancer stem cells present in a patient's cancer of atleast 85%, at least 90%, or at least 95%. Indeed, treatment of cancer inaccordance with the invention may give rise to a reduction in theproportion of cancer stem cells present in a patient's cancer of atleast 96%, at least 97%, at least 98%, at least 99%, or even 100% (suchthat substantially no cancer stem cells remain).

Asymmetric division of cancer stem cells contributes to the growth oftumours. Treatment of cancer with a ProTide compound, as defined herein,in accordance with the present invention may bring about an inhibitionof tumour growth of at least 10%, at least 20%, at least 30%, or atleast 40%. Suitably treatment of cancer in accordance with the inventionmay give rise to an inhibition of tumour growth of at least 50%, atleast 60%, at least 70%, or at least 80%. Treatment of cancer inaccordance with the invention may give rise to an inhibition of tumourgrowth of at least 85%, at least 90%, or at least 95% in a patient sotreated. Indeed, treatment of cancer in accordance with the inventionmay give rise to an inhibition of tumour growth of at least 96%, atleast 97%, at least 98%, at least 99%, or even 100% in a treated cancer.

Tumour growth may be assessed by any suitable method in which the changein size of a tumour is assessed over time. Suitably the size of a tumourprior to cancer treatment may be compared with the size of the sametumour during or after cancer treatment. A number of ways in which thesize of a tumour may be assessed are known. For example, the size of atumour may be assessed by imaging of the tumour in situ within apatient. Suitable techniques, such as imaging techniques, may allow thevolume of a tumour to be determined, and changes in tumour volume to beassessed.

As shown in the results set out in the Examples of this specification,the methods of treatment and medical uses of a ProTide compound, asdefined herein, of the invention are able not only to arrest tumourgrowth, but are actually able to bring about a reduction in tumourvolume in patients with cancers, including patients with relapsed orrefractory cancers. Suitably treatment of cancer in accordance with thepresent invention may give rise to a reduction in tumour volume of atleast 10%, at least 20%, at least 30%, or at least 40%. In suitableembodiments, treatment of cancer in accordance with the invention maygive rise to a reduction in tumour volume of at least 50%, at least 60%,at least 70%, or at least 80%. Treatment of cancer in accordance withthe invention may give rise to a reduction in tumour volume of at least85%, at least 90%, or at least 95%. Indeed, treatment of cancer inaccordance with the invention may give rise to a reduction in tumourvolume of at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%.

A reduction in tumour volume of the sort described above can becalculated with reference to a suitable control. For example in studiescarried out in vitro, or in vivo in suitable animal models, thereduction in tumour volume may be determined by direct comparisonbetween the volume of a tumour treated with a ProTide compound, asdefined herein, and the volume of a control tumour (which may beuntreated, or may have received treatment other than with a compound ofthe invention). It will be appreciated that such models requiring lackof treatment of a tumour may not be ethically acceptable in the contextof clinical trials or therapeutic management of patients, and in thiscase a reduction in tumour volume may be assessed by comparing thevolume of a treated tumour with the volume of the same tumour prior totreatment, or with a predicted volume that would have been attained bythe tumour had no treatment been administered.

The methods of treatment and medical uses of a ProTide compound, asdefined herein, may bring about a reduction in biomarkers indicative ofcancer. The reduction of such biomarkers provides a further assessmentby which effective treatment of cancer may be demonstrated. Suitableexamples of such biomarkers may be selected on the basis of the type ofcancer to be treated: in the case of gynaecological cancers CA125represents a suitable example of a biomarker, while in the case ofpancreatic or biliary cancers CA19.9 represents a suitable example of abiomarker, and in the case of colorectal cancers CEA may be a suitablebiomarker.

Suitably treatment of cancer in accordance with the present inventionmay give rise to a reduction in cancer biomarkers of at least 10%, atleast 20%, at least 30%, or at least 40%. In suitable embodiments,treatment of cancer in accordance with the invention may give rise to areduction in cancer biomarkers of at least 50%, at least 60%, at least70%, or at least 80%. Treatment of cancer in accordance with theinvention may give rise to a reduction in cancer biomarkers of at least85%, at least 90%, or at least 95%. Indeed, treatment of cancer inaccordance with the invention may give rise to a reduction in cancerbiomarkers of at least 96%, at least 97%, at least 98%, at least 99%, oreven 100%.

Beneficial effects, such as a reduction in the proportion of cancer stemcells present, reduction in tumour growth, or reduction in tumour volumeor cancer biomarkers, observed on treatment of cancer in accordance withthe present invention may be maintained for at least one month. Suitablysuch beneficial effects may be maintained for at least two months, atleast three months, at least four months, at least five months, or atleast six months. Indeed, such beneficial effects may be maintained forat least 12 months, at least 18 months, or at least 24 months. Suitablythe beneficial effects may be maintained for at least three years, atleast four years, at least five years, at least six years, at leastseven years, at least eight years, at least nine years, or for ten yearsor more.

In a suitable embodiment of the invention a ProTide compound, as definedherein, is used in a method of preventing or treating cancer or apre-malignant condition, by targeting cancer stem cells. In a suitableembodiment the invention provides the use of a ProTide compound, asdefined herein, in a method of preventing or treating cancer or apre-malignant condition, wherein the method reduces the tumourigenicityof one or more cancer stem cells. Suitably such methods may prevent theprogression of cancer, or inhibit tumour growth.

When a ProTide compound, as defined herein, is used in methods ormedical uses of the present invention to prevent or treat theprogression of a cancer, such prevention or treatment may cause thecancer progression to be slowed, delayed or stopped entirely.

The progress of a cancer is typically determined by assigning a stage tothe cancer. Staging is usually carried out by assigning a number from Ito IV to the cancer, with I being an isolated cancer and IV being acancer that has spread to the limit of what the assessment measures.Specifics of staging vary between cancers, but the stage generally takesinto account the size of a tumour, whether it has invaded adjacentorgans, how many regional (nearby) lymph nodes it has spread to (ifany), and whether it has appeared in more distant locations(metastasised).

Generally, Stage I is localised to one part of the body and may betreated by surgical resection (for solid tumours that are small enough).Stage II is locally advanced, and is treatable by chemotherapy,radiation therapy, surgery, or a combination thereof. Stage III is alsolocally advanced and the designation of Stage II or Stage III depends onthe specific type of cancer, although Stage III is generally accepted tobe “late” locally advanced. Stage IV cancers have often metastasised toa second organ. Treatment of cancer using a ProTide compound, as definedherein, in the methods or medical uses of the present invention may beused to treat a stage I, II, III or IV cancer by targeting cancer stemcells. Treatment with a ProTide compound, as defined herein, may be usedto prevent the progression of a cancer from one stage to the next. Inone embodiment, treatment with a ProTide compound, as defined herein, isused to prevent progression from Stage I to Stage II. In anotherembodiment, treatment with a ProTide compound, as defined herein, isused to prevent progression from Stage II to Stage III. In still anotherembodiment, treatment with a ProTide compound, as defined herein, isused to prevent progression from Stage III to Stage IV.

Preventing or inhibiting progression of the cancer is particularlyimportant for preventing the spread of the cancer, for example theprogression from Stage I to Stage II where the cancer spreads locally,or the progression from Stage III to Stage IV where the cancermetastasises to other organs. Cancer stem cells are tumourigenic and soare believed to play a critical role in the spread of cancer, bothlocally and metastatically. Methods of treatment or medical uses of theinvention employing a ProTide compound, as defined herein, can thereforebe used to prevent the spread of cancer, by targeting tumourigeniccancer stem cells and thus reducing their numbers.

“Cancers”

The ProTide compounds that are the subject of the present inventiondemonstrate beneficial anti-cancer activity, and some demonstrateincreased anti-cancer activity as compared to the compounds from whichthey are derived. The anti-cancer activity exhibited by these ProTidecompounds includes activity that is observed in respect of both cancerstem cells and non-stem cancer cells.

Cancer stem cells play a role in the biological activity of a wide rangeof cancers. Accordingly, there are a wide range of cancers that may beprevented or treated in accordance with the present invention.

As discussed elsewhere herein, cancer stem cells are known to be presentin many tumour types including liquid tumours (including haematologicaltumours such as leukaemias and lymphomas) and solid tumours (such asbreast, lung, colon, prostate, ovarian, skin, bladder, biliary andpancreas tumours). Methods of treatment and medical uses of a ProTidecompound, as defined herein, to target cancer stem cells are thereforeexpected to be useful in the prevention or treatment of such cancers.

Suitably a ProTide compound, as defined herein, may be used in theprevention or treatment of a cancer selected from the group consistingof: leukaemia, lymphoma, multiple myeloma, lung cancer, liver cancer,breast cancer, head and neck cancer, neuroblastoma, thyroid carcinoma,skin cancer (including melanoma), oral squamous cell carcinoma, urinarybladder cancer, Leydig cell tumour, biliary cancer, such ascholangiocarcinoma or bile duct cancer, pancreatic cancer, colon cancer,colorectal cancer and gynaecological cancers, including ovarian cancer,endometrial cancer, fallopian tube cancer, uterine cancer and cervicalcancer, including epithelia cervix carcinoma. In suitable embodiments,the cancer is leukaemia and can be selected from the group consisting ofacute lymphoblastic leukaemia, acute myelogenous leukaemia (also knownas acute myeloid leukaemia or acute non-lymphocytic leukaemia), acutepromyelocytic leukaemia, acute lymphocytic leukaemia, chronicmyelogenous leukaemia (also known as chronic myeloid leukaemia, chronicmyelocytic leukaemia or chronic granulocytic leukaemia), chroniclymphocytic leukaemia, monoblastic leukaemia and hairy cell leukaemia.In further preferred embodiments, the cancer is acute lymphoblasticleukaemia. In a suitable embodiment the cancer is lymphoma, which may beselected from the group consisting of: Hodgkin's lymphoma; non-Hodgkinlymphoma; Burkitt's lymphoma; and small lymphocytic lymphoma.

Suitably targeting cancer stem cells in such cancers may achieveeffective treatment of the cancer by preventing or treating thedevelopment of the cancer, by preventing or treating the progression ofthe cancer, by preventing or treating the recurrence of the cancer, orby preventing or treating the propagation of the cancer.

In a suitable embodiment the present invention provides a ProTidecompound, as defined herein, for use in targeting cancer stem cells inthe prevention or treatment of metastatic cancer.

In a suitable embodiment the present invention provides a ProTidecompound, as defined herein, for use in targeting cancer stem cells inthe treatment of relapsed or refractory cancer.

In a suitable embodiment the present invention provides a ProTidecompound, as defined herein, for use in targeting cancer stem cells inthe treatment of a primary cancer. Suitably the primary cancer treatedmay be a second primary cancer.

The invention provides a ProTide compound, as defined herein, for use intargeting cancer stem cells in the treatment of secondary cancer. In asuitable embodiment the secondary cancer is a metastatic cancer.

In a suitable embodiment the present invention provides a ProTidecompound, as defined herein, for use in targeting cancer stem cells,wherein the targeting of cancer stem cells prevents or inhibits: (i)recurrence of a cancer; (ii) occurrence of second primary cancer; or(iii) metastasis of a cancer.

Methods of treatment or medical uses in which a ProTide compound, asdefined herein, is employed on the basis of its ability to target cancerstem cells may be used in the treatment of relapsed or refractorycancer. The considerations regarding relapsed or refractory cancer insuch embodiments are, except for where the context requires otherwise,the same as for the treatment of relapsed or refractory cancer inconnection with the other aspects or embodiments of the invention.

“Relapsed or Refractory Cancer”

As noted above, certain aspects and embodiments of the inventionparticularly relate to the use of a ProTide compound, as defined herein,in the treatment of relapsed or refractory cancers.

For the purposes of the present invention, refractory cancers may betaken as cancers that demonstrate resistance to treatment by anti-cancertherapies other than those utilising a compound of the invention. Forexample, a ProTide compound, as defined herein, may be used in thetreatment of refractory cancers that are resistant to treatment withradiotherapy. Alternatively, or additionally, a ProTide compound, asdefined herein, may be used in the treatment of refractory cancers thatare resistant to biological agents used in the treatment of cancer. In asuitable embodiment a ProTide compound, as defined herein, may be usedin the treatment of refractory cancers that are resistant to treatmentwith chemotherapeutic agents other than a compound of the invention.

In particular, refractory cancers that may benefit from the methods oftreatment of medical uses of the invention employing a ProTide compound,as defined herein, include those cancers that are resistant to parentcompounds from which the various ProTides are derived (for examplecancers resistant to FUDR, thioguanosine, cladribine, thioinosine,fludarabine, or clofarabine).

Relapsed cancers (or recurrent cancers) are those that return after aperiod of remission during which the cancer cannot be detected. Cancerrecurrence may occur at the site of the original cancer (local cancerrecurrence), at a site close to that of the original cancer (regionalcancer recurrence), or at a site distant from that of the originalcancer (distal cancer recurrence). Cancer stem cells are believed toplay a role in the recurrence of cancer, providing a source from whichcells of the relapsed cancer are generated. Accordingly, the methods oftreatment and medical uses of a ProTide compound, as defined herein, inaccordance with the invention, which enable targeting of cancer stemcells, may be of great benefit in the context of relapsed cancers. Theability of a ProTide compound, as defined herein, to target cancer stemcells may be used to remove the populations of such cells that are ableto give rise to recurrence, thus preventing incidences of relapsedcancer. The anti-cancer stem cell activity of a ProTide compound, asdefined herein, may also be used to target cancer stem cells in cancersthat have recurred, as well as potentially exerting cytotoxic effects onnon-stem cancer cells, thereby providing treatment of relapsed cancers.

In view of the above, it will be appreciated that a ProTide compound, asdefined herein, may be used in the methods or uses of the invention forthe prevention or treatment of a relapsed cancer. A ProTide compound, asdefined herein, may be used in the methods or uses of the invention forthe prevention or treatment of a local, regional or distant relapsedcancer.

A ProTide compound, as defined herein, may be used in the methods oruses of the invention to prevent the recurrence of cancer by providingat least 2 months, at least 6 months, at least 12 months, at least 18months, at least 24 months, or at least 30 months of remission. Indeed,a ProTide compound, as defined herein, may be used to prevent recurrenceof cancer by providing at least 4 years, at least 5 years, at least 6years, at least 7 years, at least 8 years, at least 9 years, or at least10 years of remission.

A ProTide compound, as defined herein, may be used in the methods oruses of the invention to treat a relapsed cancer which has recurredafter at least 2 months, at least 6 months, at least 12 months, at least18 months, at least 24 months, or at least 30 months of remission.Indeed, a ProTide compound, as defined herein, may be used to treat arelapsed cancer which has recurred after at least 4 years, at least 5years, at least 6 years, at least 7 years, at least 8 years, at least 9years, or at least 10 years of remission.

The ability of the compounds of the invention to target cancer stemcells gives rise to the ability of these compounds to prevent or treatcancers in accordance with the medical uses or methods of treatment ofthe invention. However, it should be noted that compounds of theinvention also exert a direct cytotoxic effect upon non-stem cancercells that make up the bulk of tumours. While activity of cancer stemcells may underlie much of the resistance that makes relapsed orrefractory cancers so difficult to treat, non-stem cancer cells are alsoa major constituent of such relapsed or refractory cancers.

Compounds of the invention may exert greater cytotoxic effects onnon-stem cancer cells than do the chemotherapeutic molecule from whichthe compounds of the invention are derived. Accordingly, the mechanismby which a ProTide compound, as defined herein, acts in the treatment ofrelapsed or refractory cancer may not be limited solely to theanti-cancer stem cell activity of this compound, but may also make useof the action of a ProTide compound, as defined herein, on non-stemcancer cells. In such uses treatment with a ProTide compound, as definedherein, will reduce the total number of both cancer stem cells andnon-stem cancer cells, but will preferentially reduce the proportion ofcancer stem cells that remain after treatment.

Therapeutically Effective Doses of a Compound of the Invention

A therapeutically effective amount of a ProTide compound, as definedherein, may be an amount sufficient to induce death of cancer cells. Atherapeutically effective amount of a ProTide compound, as definedherein, may be an amount sufficient to induce death of cancer stemcells. In some embodiments, particularly those relating to the treatmentof relapsed or refractory cancer, a therapeutically effective amount ofa ProTide compound, as defined herein, may be an amount sufficient toinduce death of cancer stem cells and also to induce death of non-stemcancer cells.

There are various different ways in which the amount of atherapeutically effective compound, such as a compound of the invention,to be administered to a patient may be calculated and expressed. Onesuch way which is considered particularly relevant in doses of agentsfor the prevention or treatment of cancer, is in the amount of the agentto be administered per unit of body surface area of the patient. Suchdoses are typically expressed in terms of the amount of the agent (whichmay be determined by mass) per square meter (m²) of surface area.

Uses of a ProTide compound, as defined herein, for the prevention ortreatment of cancer may utilise a weekly dose of between 250 mg/m² and1000 mg/m². Such treatments may, for example utilise a weekly dose ofbetween 375 mg/m² and 900 mg/m². For example, effective treatment ofrelapsed or refractory cancers may be provided when patients areprovided with weekly doses of a ProTide compound, as defined herein,that range between approximately 500 mg/m² and 825 mg/m².

Without wishing to be bound by any hypothesis, the inventors believethat the ability of a ProTide compound, as defined herein, to targetcancer stem cells allows therapeutic effectiveness to be achieve usinglower doses of this compound than would otherwise be expected. Merely byway of example, weekly doses of a ProTide compound, as defined herein,that are as low as 825 mg/m², 750 mg/m², 600 mg/m², or 500 mg/m² mayprove therapeutically effective in the uses and methods of theinvention.

A chosen weekly dose of a ProTide compound, as defined herein, may beprovided in a single incidence of administration, or in multipleincidences of administration during a week. For example, a weekly doseof a ProTide compound, as defined herein, may be provided in twoincidences of administration, in three incidences of administration, ormore. Thus, in the case of a weekly dose of 750 mg/m², this may beachieved by three administrations of 250 mg/m² over the course of aweek, or two administrations of 375 mg/m² during a week Similarly, inthe case of a weekly dose of 600 mg/m², this may be achieved by threeadministrations of 200 mg/m² over the course of a week, or twoadministrations of 300 mg/m² during a week.

A suitable amount of a ProTide compound, as defined herein, to beadministered in a single incidence of treatment in order to provide arequired dose of this compound over the course of week may be betweenapproximately 100 mg/m² and 300 mg/m².

The weekly dose of a ProTide compound, as defined herein, provided maydecrease over the course of treatment. For example, treatment may bestarted at a weekly dose of around 1000 mg/m², 900 mg/m², 825 mg/m², 750mg/m², or 725 mg/m², and over the course of treatment the dose neededmay decrease to around 750 mg/m² (in cases where the initial dose isabove this amount), around 650 mg/m², around 625 mg/m², or even around500 mg/m² or around 375 mg/m².

Doses of a ProTide compound, as defined herein, can, of course, bepresented in other manners. The most common of these is the amount ofthe active agent to be provided per unit body mass. It has beencalculated that for an average human patient a dose of 1 mg/m² isequivalent to approximately 0.025 mg/kg body mass. Accordingly, the dataindicate that a ProTide compound, as defined herein, is effective forthe treatment of relapsed or refractory cancer at doses ranging fromapproximately 6.25 mg/kg to approximately 25 mg/kg. A suitable dose may,for example, be of between about 9.5 mg/kg and 22.5 mg/kg. In a suitableembodiment a ProTide compound, as defined herein, achieves effectivetreatment of relapsed or refractory cancers when patients are providedwith weekly doses ranging between approximately 12.5 mg/kg and 20.5mg/kg.

Considerations regarding formulations of a ProTide compound, as definedherein, suitable for use in the methods of prevention or treatment andmedical uses of the present invention are described elsewhere in thisdisclosure. In the case of injectable formulations of a compound of theinvention, these may be administered intravenously. Intravenousadministration may be achieved over any suitable time frame, for examplein a ten minute injection, or the like.

Types of Treatment

In a suitable embodiment a ProTide compound, as defined herein, may beused for targeting cancer stem cells as a first line treatment ofcancer.

However, the finding that compounds of the invention are able to targetcancer stem cells and thereby treat relapsed or refractory cancerillustrates that a ProTide compound, as defined herein, is able toprovide effective treatment of cancer in contexts in which othertreatments have proved ineffective. Accordingly, in a suitableembodiment the present invention provides a ProTide compound, as definedherein, for targeting cancer stem cells as a second line treatment ofcancer. Indeed, in a suitable embodiment the present invention providesa ProTide compound, as defined herein, for targeting cancer stem cellsas a third, or further, line treatment of cancer.

In a suitable embodiment there is provided a ProTide compound, asdefined herein, for use as a neoadjuvant in the treatment of cancer. Aneoadjuvant is an agent provided to a patient in order to reduce thesize of a tumour prior to a “main” anti-cancer therapy, such as surgicalremoval of cancer. A ProTide compound, as defined herein, may be used asa neoadjuvant therapy for a patient who will subsequently undergosurgical treatment of cancer and/or radiotherapy for cancer.

Alternatively, or additionally, the invention provides a ProTidecompound, as defined herein, for use as an adjuvant in the treatment ofcancer. An adjuvant is an agent provided to a patient after a “main”anti-cancer therapy, such as surgical removal of cancer, in order toprevent the return of cancer after the main therapy. A ProTide compound,as defined herein, may be used as an adjuvant for a patient who hasundergone surgical treatment of cancer and/or radiotherapy for cancer.

A ProTide compound, as defined herein, may be employed in the methods oruses of the invention in a monotherapy, which is to say in preventionsor treatments in which a ProTide compound, as defined herein, providessubstantially all of the therapeutic activity that is made use of in theprevention or treatment.

Alternatively, the methods or uses of the invention may employ a ProTidecompound, as defined herein, in a combination therapy. In suchembodiments a ProTide compound, as defined herein, is used inconjunction with at least one further cancer therapy. The further cancertherapy may comprise surgery and/or radiotherapy. Additionally, oralternatively, the further cancer therapy may comprise use of at leastone further therapeutic agent that contributes to the prevention ortreatment of cancer to be achieved. Suitably such an agent may be achemotherapeutic agent or a biological agent used in the prevention ortreatment of cancer.

In a suitable embodiment of a combination therapy a ProTide compound, asdefined herein, and a further therapeutic agent may be provided to apatient at the same time. In a suitable example, the ProTide compound,as defined herein, and a further therapeutic agent may be formulated aspart of the same pharmaceutical composition. Alternatively the ProTidecompound, as defined herein, and a further therapeutic agent may beformulated separately for provision to the patient at substantially thesame time.

n another suitable embodiment of a combination therapy, a ProTidecompound, as defined herein, and a further therapeutic agent may beprovided to a patient at different times. The ProTide compound, asdefined herein, and a further therapeutic agent may be provided to apatient sequentially. For example, the ProTide compound, as definedherein, may be provided to the patient prior to provision of the furthertherapeutic agent. Alternatively a ProTide compound, as defined herein,may be provided to the patient after provision of the furthertherapeutic agent.

“Further Therapeutic Agents”

A ProTide compound, as defined herein, may be used in combination with awide range of further therapeutic agents for the prevention or treatmentof cancer. These include biological agents, immunotherapeutic agents,and chemotherapeutic agents that may be used for the prevention ortreatment of cancer.

While specific examples of suitable further agents are considered in thefollowing paragraphs, these should not be taken as limiting the range offurther therapeutic agents suitable for use with a compound of theinvention. Indeed, the ability of a ProTide compound, as defined herein,to target cancer stem cells indicates that it may be beneficially usedin combination with any further therapeutic agent used in the preventionor treatment of cancer, whether such further agent targets cancer stemcells, non-stem cancer cells, or other cells or constituents involved inthe development, maintenance, recurrence, propagation of cancer.

Examples of further therapeutic agents that may be used in combinationwith a ProTide compound, as defined herein, include:

(a) an anti-angiogenic agent, optionally wherein the anti-angiogenicagent is: (i) an inhibitor of the VEGF pathway, optionally bevacizumab;(ii) a tyrosine kinase inhibitor, optionally sorafenib, sunitinib orpazopanib; or (iii) an mTOR inhibitor, optionally everolimus;(b) an alkylating agent;(c) an anti-metabolite;(d) an anti-tumour antibiotic;(e) a topoisomerase;(f) a mitotic inhibitor;(g) a monoclonal antibody;(h) a metallic agent; or(i) an active or passive immunotherapy.

Except for where the context requires otherwise, the further therapeuticagents set out in the preceding list should all be considered suitablefor use in any of the embodiments of combination therapies with aProTide compound, as defined herein, considered above.

Selection of Patients

The inventors' finding that a ProTide compound, as defined herein, isable to target cancer stem cells makes possible a number of methods bywhich it is possible to determine whether a particular patient is likelyto benefit from receiving a ProTide compound, as defined herein, in theprevention or treatment of cancer, such as relapsed or refractorycancer.

Accordingly, the invention provides a method of determining whether apatient with cancer or a pre-cancerous condition will benefit fromprevention or treatment of cancer with a compound of the invention, themethod comprising: assaying a biological sample representative of canceror a pre-cancerous condition in the patient for the presence of cancerstem cells; wherein the presence of cancer stem cells in the biologicalsample indicates that the patient will benefit from treatment with acompound of the invention.

The invention further provides a method of determining a suitabletreatment regimen for a patient with cancer or a pre-cancerouscondition, the method comprising: assaying a biological samplerepresentative of cancer or a pre-cancerous condition in the patient forthe presence of cancer stem cells; wherein the presence of cancer stemcells in the biological sample indicates that a suitable treatmentregimen will comprise treatment of the patient with a compound of theinvention.

The invention also provides a ProTide compound, as defined herein, foruse in the prevention or treatment of cancer in a patient selected forsuch treatment by a method comprising: assaying a biological samplerepresentative of cancer or a pre-cancerous condition in the patient forthe presence of cancer stem cells; wherein the presence of cancer stemcells in the biological sample indicates that the patient is suitablefor treatment with a compound of the invention.

In suitable embodiments cancer stem cells in a biological sample may beidentified by their expression of characteristic patterns of markersdiscussed previously in the application.

The skilled person will appreciate that there are many suitable examplesof biological samples that may be used in embodiments of the inventionsuch as those set out above. Suitably such a sample may include cellsfrom the cancer or pre-cancerous condition. A suitable biological samplemay be a tissue sample, such as a sample for use in histology. Cells insuch samples may be directly assessed for their expression of cancerstem cell markers, such as those set out above.

Alternatively or additionally, a suitable biological sample may comprisetarget molecules representative of gene expression by cells of thecancer or pre-cancerous condition. Examples of such target moleculesinclude proteins encoded by the genes expressed, or nucleic acids, suchas mRNA, representative of gene expression.

Suitable examples of techniques by which expression of cancer stem cellmarkers may be assessed may be selected with reference to the sampletype. Techniques for the investigation of expressed markers arefrequently used in the context of clinical assessments (such as fordiagnostic or prognostic purposes) and their use will be familiar tothose required to practice them in the context of the present invention.Merely by way of example, in samples containing proteins the presence ofcancer stem cell markers may be assessed by suitable techniques usingantibodies that react with the cancer stem cell markers in question.Examples of such samples containing protein cancer stem cell markersinclude histology samples (where the presence of the markers may bevisualised by suitable immunocytochemistry techniques), or samplesderived from the circulation. Here the presence of circulating cancerstem cells (which are believed to contribute to the propagation ofcancer through metastasis) may be assessed using techniques such as flowcytometry.

In samples containing nucleic acids representative of expression ofcancer stem cell markers, such expression may be assessed by suitablemolecule biology techniques, such as by polymerase chain reaction (PCR)amplification using suitable primers.

Synthesis

The ProTides discussed herein can be synthesised according to oranalogously to the methods described in WO 2005/012327, WO 2012/117246and WO2006/100439.

The invention will now be further described with reference to thefollowing Examples, and the accompanying drawings in which:

FIG. 1A illustrates the results of investigation of potency of theanti-cancer agent thioinosine, and of ProTide compounds derived fromthis agent. FIG. 1A compares LD₅₀ values for thioinosine, CPF 761 andCPF 762. All assays were carried out using KG1a cells and data arepresented as mean (±SD) of five independent experiments. FIG. 1Billustrates the results of studies investigating the ability of thesecompounds to target cancer stem cells. FIG. 1B provides analysis ofleukaemia stem cell targeting capacity of thioinosine and ProTides CPF761 and CPF 762. All data are the mean (±SD) of three independentexperiments.

FIG. 2A illustrates the results of investigation of potency of theanti-cancer agent clofarabine, and of ProTide compounds derived fromthis agent. FIG. 2A compares the compares LD₅₀ values for clofarabine,CPF 448, CPF 720 and CPF 727. All assays were carried out using KG1acells and data are presented as mean (±SD) of five independentexperiments. FIG. 2B illustrates the results of studies investigatingthe ability of these compounds to target cancer stem cells.

FIG. 3A illustrates the results of investigation of potency of theanti-cancer agent clofarabine, and of a ProTide compound (CPF 727) andisomers (A and B) of the ProTide compound CPF 448) derived from thisagent, and FIG. 3B illustrates the results of studies investigating theability of these compounds to target cancer stem cells.

FIG. 4A illustrates the results of investigation of potency of theanti-cancer agent cladribine, and of ProTide compounds derived from thisagent. FIG. 4A compares the compares LD₅₀ values for cladribine, CPF 791and CPF 793. All assays were carried out using KG1a cells and data arepresented as mean (±SD) of five independent experiments. FIG. 4Billustrates the results of studies investigating the ability of thesecompounds to target cancer stem cells. FIG. 4B provides analysis ofleukaemia stem cell targeting capacity of cladribine and ProTides CPF791 and CPF 793. All data are the mean (±SD) of three independentexperiments.

FIG. 5A illustrates the results of investigation of potency of theanti-cancer agent fludarabine, and of ProTide compounds derived fromthis agent. FIG. 5A compares the compares LD₅₀ values for fludarabine,CPF 682 and CPF 544. All assays were carried out using KG1a cells anddata are presented as mean (±SD) of five independent experiments. FIG.5B illustrates the results of studies investigating the ability of thesecompounds to target cancer stem cells. FIG. 5B provides analysis ofleukaemia stem cell targeting capacity of fludarabine, CPF 682 and CPF544. All data are the mean (±SD) of three independent experiments.

FIG. 6A illustrates the results of investigation of potency of theanti-cancer agent thioguanosine, and of ProTide compounds derived fromthis agent. FIG. 6A compares the compares LD₅₀ values for thioguanosine,and ProTides CPF 775 and CPF 782. All assays were carried out using KG1acells and data are presented as mean (±SD) of five independentexperiments. FIG. 6B illustrates the results of studies investigatingthe ability of these compounds to target cancer stem cells. FIG. 6Bprovides analysis of leukaemia stem cell targeting capacity ofthioguanosine, and ProTides CPF 775 and CPF 782. All data are the mean(±SD) of three independent experiments.

FIG. 7A illustrates the results of investigation of potency of theanti-cancer agent FUDR, and of ProTide compounds derived from thisagent. FIG. 7A compares the compares LD₅₀ values for 5-FU, FUDR andProTides CPF 373 (mixture) and its purified isomers, CPF 585, CPF 381,and CPF 581. All assays were carried out using KG1a cells and data arepresented as mean (±SD) of five independent experiments. FIG. 7Billustrates the results of studies investigating the ability of thesecompounds to target cancer stem cells. FIG. 7B provides analysis ofleukaemia stem cell targeting capacity of 5-FU, FUDR and ProTides CPF373 (mixture) and its purified isomers, CPF 585, CPF 381, and CPF 581.All data are the mean (±SD) of three independent experiments.

FIG. 8A illustrates the results of investigation of potency of theanti-cancer agent gemcitabine, and of the comparator ProTide compoundNUC-1031 derived from this agent. FIG. 8A compares the compares LD₅₀values for gemcitabine, ProTide NUC-1031 and the purified Rp-andSp-isomers. FIG. 8B illustrates the results of studies investigating theability of these compounds to target cancer stem cells. FIG. 8B providesanalysis of leukaemia stem cell targeting capacity of gemcitabine andNUC-1031 ProTides. All data are the mean (±SD) of three independentexperiments.

EXAMPLES AND COMPARATIVE EXAMPLES 1 Preferential Targeting of CancerStem Cells by ProTide Compounds

The following study illustrates the ability of ProTide compounds, asdefined herein, to preferentially target CSCs in vitro, and therebyreduce the proportion of CSCs present in populations of cancer cells.The cancer stem cell targeting ability of ProTide compounds encompassedby the present invention is compared with that of the comparator ProTidecompound, NUC-1031.

1.1 Comparison of LD₅₀ Values for ProTide Compounds and the ParentCompounds from which they are Derived in Primary Acute Myeloid LeukaemiaBlasts

ProTide compounds, as defined herein, and the comparator ProTidecompound NUC-1031 were assayed for their cytotoxic effects on primarycultures of acute myeloid leukaemia (AML) blasts. LD₅₀ values (theconcentration required to kill 50% of the tumour cells in culture) werecalculated in respect of the ProTide compounds of the invention, and theparent compounds from which they are derived. As can be seen (set outbelow) certain of the ProTide compounds exhibited greater potency thanthe parent compounds from which they were derived. The LD₅₀ values ofthe ProTide compounds as defined herein (and their parent compounds)were compared with the comparator ProTide NUC-1031 and its parentcompound gemcitabine.

Parent compound ProTide compound CPF 581 0.027 μM FUDR 1.18 μM CPF 3810.039 μM FUDR 1.18 μM CPF 373 (mix) 0.053 μM FUDR 1.18 μM CPF 373 (IsoA) 0.052 μM FUDR 1.18 μM CPF 373 (Iso B) 0.055 μM FUDR 1.18 μM CPF 5850.79 μM FUDR 1.18 μM CPF 782 0.82 μM Thioguanosine 1 μM ComparatorProTide compound NUC-1031 0.28 μM Gemcitabine 1.4 μM

In Vitro Cytotoxicity Assay in Primary Acute Myeloid Leukaemia Cells

Bone marrow samples were collected in ethylenediaminetetraacetic acid(EDTA) from newly diagnosed, previously untreated, acute myeloidleukaemia (AML) patients. AML blasts were enriched by density gradientcentrifugation using Histopaque (Sigma, Poole, UK) and were subsequentlymaintained in Roswell Park Memorial Institute medium (RPMI) supplementedwith 10% foetal bovine serum (FBS). Cells were treated with ProTidecompounds (either as encompassed by the present invention, or thecomparator ProTide NUC-1031) or with the ProTides' parent compounds at arange of experimental concentrations, and incubated for 48 h. Allcultures were maintained at 37° C. in a 5% CO₂ humidified atmosphere.

Measurement of In Vitro Apoptosis in Primary AML Cells

Cells were harvested and labelled with CD34-fluorescein isothiocyanate(FITC) (BD Biosciences, Buckingham, UK) and then resuspended in 200 μlof binding buffer containing 4 μl of annexin V labelled withallophycocyanin (APC) (eBioscience Ltd, Hatfield, UK). Apoptosis wasquantified in the CD34⁺ AML cells using an Accuri C6 flow cytometer(Becton Dickinson, CA, USA). At least 10,000 events were acquired anddata were subsequently analysed using FlowJo software (Tree Star Inc.,Ashland, OR, USA). All LD₅₀ values (concentration of drug required tokill 50% of cells) were derived from the dose-response curves.

Identification and Quantification of CD34⁺/CD123⁺ Sub-Populations inPrimary AML Cells Putative leukaemic stem cells were identified by dualexpression of CD34 and CD123. The relative sensitivity of these cells tothe effects of ProTides and their parent compounds were assessed as afunction of the percentage of these cells that remained viable followingexposure to molar equivalents of each agent.

The results of this study allowed the calculation of mean LD₅₀ valueobtained in respect of both ProTide compounds and the parent anti-canceragents from which they are derived. Increased potency of ProTidederivatives was indicated LD₅₀ values lower than those of their parentcompound, and the results obtained in this study are discussed furtherbelow.

The ability of ProTide compounds and their parent anti-cancer agents todeplete CSCs in the AML blast cultures at a range of experimentalconcentrations was also investigated, and the results reported below.The ability of a ProTide or parent compound to target stem cells isdemonstrated by a reduction in the proportion of cancer stem cellspresent in a population of treated cells.

1.2 Preferential Targeting of CSCs by NUC-1031

The respective abilities of NUC-1031 and gemcitabine to target CSCs wereinvestigated in the AML cell line KG1a. The KG1a cell line was chosen inparticular for this study because CSCs within the population exhibit aLin⁻/CD34⁺/CD38⁻/CD123⁺ immunophenotype that allows them to readily bedistinguished from non-stem cancer cells (also termed “bulk” cancercells) within the population.

1.3 KG1a Cell Culture Conditions

The acute myeloid leukaemia (AML) KG1a cell line was maintained in RPMImedium (Invitrogen, Paisley, UK) supplemented with 100 units/mlpenicillin, 100 μg/ml streptomycin and 20% fetal calf serum. Cells weresubsequently aliquoted (10⁵ cells/100p) into 96-well plates and wereincubated at 37° C. in a humidified 5% carbon dioxide atmosphere for 72h in the presence of NUC-1031 or gemcitabine at concentrations that wereexperimentally determined for each compound. In addition, controlcultures were carried out to which no drug was added. Cells weresubsequently harvested by centrifugation and were analysed by flowcytometry using the Annexin V assay.

1.4 Measurement of In Vitro Apoptosis

Cultured cells were harvested by centrifugation and then resuspended in195 μl of calcium-rich buffer. Subsequently, 5 μl of Annexin V (CaltagMedsystems, Botolph Claydon, UK) was added to the cell suspension andcells were incubated in the dark for 10 mins prior to washing. Cellswere finally resuspended in 190 μl of calcium-rich buffer together with10 μl of propidium iodide. Apoptosis was assessed by dual-colourimmunofluorescent flow cytometry as described previously. SubsequentlyLD₅₀ values (the dose required to kill 50% of the cells in a culture)were calculated for each nucleoside analogue and ProTide.

1.5 Immunophenotypic Identification of the Leukaemic Stem CellCompartment

KG1a cells were cultured for 72 hours in the presence of a wide range ofconcentrations of each nucleoside analogue and their respectiveProTides. Cells were then harvested and labelled with a cocktail ofanti-lineage antibodies (PE-cy7), anti-CD34 (FITC), anti-CD38 (PE) andanti-CD123 (PERCP cy5). The sub-population expressing a leukaemic stemcell (LSC) phenotype were subsequently identified and were expressed asa percentage of all viable cells left in the culture. The percentages ofstem cells remaining were then plotted on a dose-response graph and theproportion of cancer stem cells was compared across the variousconcentrations of ProTides and their respective parent compounds, inorder to determine if they preferentially target cancer stem cells.

1.6 Statistical Analysis

The data obtained in these experiments were evaluated using one wayANOVA. All data was confirmed as Gaussian or a Gaussian approximationusing the omnibus K2 test. LD₅₀ values were calculated from thenon-linear regression and line of best-fit analysis of the sigmoidaldose-response curves. All statistical analyses were performed usingGraphpad Prism 6.0 software (Graphpad Software Inc., San Diego, Calif.).

1.7 Thioinosine and ProTide Derivatives of Thioinosine

ProTide derivatives of Thioinosine exhibited reduced potency as comparedto the parent compound (FIG. 1A).

In KG1 a cells, treatment with Thioinosine, CPF-761 or CPF-762 resultedin a reduction of the proportion of cancer stem cells as compared tonon-stem cancer cells (FIG. 1 ), suggesting that Thioinosine, CPF-761and CPF-762 preferentially target cancer stem cells.

Additionally, CPF-761 and CPF-762 showed greater selectivity againstcancer stem cells than Thioinosine. Of the three compounds, CPF-761appeared to be the most selective against cancer stem cells, and theincrease in selectivity was statistically significant as compared to theparent compound.

1.8 Clofarabine and ProTide Derivatives of Clofarabine

CPF-727, a ProTide derivative of clofarabine exhibited potency that didnot significantly differ from that of the parent compound, whereasProTides CPF-448 and CPF-720 both exhibited reduced potency as comparedto the parent compound (FIG. 2A). Further investigation of theproperties of Isomers A and B of CPF-448 indicated that both of thesewere significantly less potent than the parent compound, but that IsomerA was significantly more potent than Isomer B.

In KG1a cells, treatment with Clofarabine, CPF-448, CPF-720 or CPF-727showed a reduction in the proportion of cancer stem cells as compared tonon-stem cancer cells (FIG. 2A). This suggests that Clofarabine,CPF-448, CPF-720 and CPF-727 also preferentially target cancer stemcells. This selective effect was observed even at a very lowconcentration of approximately 6×10⁻⁸ M. It should also be noted thatCPF-448 showed greater selectivity against cancer stem cells thanClofarabine itself.

When the ability of the different isomers of CPF-448 to selectivelytarget cancer stem cells was investigated, Isomer A proved to be moreselective than Isomer B, and only Isomer A showed a statisticallysignificant increase in selectivity. This increase in selectivity wasnoted both in comparison to the parent compound, and ProTide compoundCPF-727 (FIG. 3B).

1.9 Cladribine and ProTide Derivatives of Cladribine

ProTide derivatives of cladribine exhibited reduced potency as comparedto the parent compound (FIG. 4A).

In KG1a cells, treatment with Cladribine, CPF-791 or CPF-793, resultedin a reduction in the proportion of cancer stem cells as compared tonon-stem cancer cells (FIG. 4B), suggesting that these three compoundsalso preferentially target cancer stem cells. The reduction inproportion of cancer stem cells was particularly striking in cellstreated with CPF-793. The selectivity of this compound was observed evenat a very low concentration (approximately 6×10⁻⁸ M).

Additionally, both ProTide derivatives (CPF-791 and CPF-793) showed agreater selectivity against cancer stem cells than the parentcompound—Cladribine. This increase in selectivity was statisticallysignificant in respect of ProTide CPF-793.

1.10 Fludarabine and ProTide Derivatives of Fludarabine

ProTide derivatives of fludarabine exhibited reduced potency as comparedto the parent compound (FIG. 5A).

In KG1a cells, treatment with Fludarabine, CPF-544 or CPF-682 resultedin a significant decrease in the proportion of cancer stem cells ascompared to non-stem cancer cells (FIG. 5B). This shows thatFludarabine, CPF-544 or CPF-682 preferentially target cancer stem cells.The selectivity of the ProTide derivatives (CPF-544 and CPF-682) againstcancer stem cells was found to be comparable to that of Fludarabine.

1.11 Thioguanosine and ProTide Derivatives of Thioguanosine

ProTide derivatives of thioguanosine exhibited reduced potency ascompared to the parent compound (FIG. 6A).

In KG1a cells, treatment with Thioguanosine, CPF-782 or CPF-775 showed astrong reduction in the proportion of cancer stem cells as compared tonon-stem cancer cells (FIG. 6B). This indicates that all three compoundspreferentially target cancer stem cells. Of these compounds, CPF-782 wasthe most selective, but both CPF-782 and CPF-775 exhibited statisticallysignificant increases in selectivity at certain concentrations.

1.12 FUDR and ProTide Derivatives of FUDR

When the in vitro potency of FUDR and ProTides CPF-585, CPF-373 (Iso A),CPF-373 (Iso B), CPF-381 or CPF-581 (all derived from FUDR) was studied,it was found that all of the ProTide derivatives other than CPF-585 havesignificantly greater potency than FUDR (FIG. 7A). For example, the LD₅₀value of CPF-581 was found to be 0.027 μM, compared to the LD₅₀ value ofFUDR, equal to 1.18 μM, indicating that CPF-581 is over 50 times morepotent than FUDR.

In KG1a cells, treatment with FUDR, CPF-585, CPF-373 (Iso A), CPF-373(Iso B), CPF-381 or CPF-581 showed a reduction in the proportion ofcancer stem cells as compared to non-stem cancer cells, indicating thatthe treatments preferentially target cancer stem cells (FIG. 7B). Thereduction in cancer stem cells was especially significant in cellstreated with CPF-373 (Iso A), CPF-373 (Iso B), CPF-381 or CPF-581, wherethe proportion of cancer stem cell numbers was reduced even in cellstreated at a very low concentration of approximately 8×10⁻⁸ M. It shouldalso be noted that all of the above mentioned ProTide derivatives ofFUDR, showed a greater selectivity against cancer stem cells than FUDR,with the increase in selectivity being statistically significant in thecase of the isoforms of CPF-373.

1.13 Comparative Example: NUC-1031 Preferentially Targets Cancer StemCells

In KG1a cells, NUC-1031 (and its purified isomers) showed increased invitro potency when compared to Gemcitabine (FIG. 8A). However, there wasno significant difference in potency between the unseparated mixture andthe purified isomers of NUC-1031.

NUC-1031 showed preferential targeting of CSCs when compared withGemcitabine. This was consistently observed at sub-micromolarconcentrations of ProTide (FIG. 8B). Again, the two purified isomers ofNUC-1031 showed no significant difference in their ability to targetCSCs in our experimental system.

It can be seen that the biological activity of NUC-1031 in vitro closelyresembles that observed in respect of a number of the other ProTidecompounds referred to above. Accordingly, the inventors have soundreasons to believe that these ProTide compounds will also share theclinical efficacy of NUC-1031, which is discussed further in thefollowing comparative Example.

2 Comparative Clinical Example: NUC-1031 is Able to Treat Relapsed orRefractory Cancers in Human Patients

The following data were generated in clinical studies of NUC-1031 inhuman patients with advance progressive cancers that are refractory to,or have relapsed on, all conventional therapies that have been used todate. The results clearly illustrate the ability of NUC-1031 tosuccessfully treat refractory cancers.

Although the primary objectives of the dose escalation part of the studywere to determine the Recommended Phase II Dose (RP2D) and safetyprofile, secondary objectives included determining the PK profile,however effective treatment of patients with a range of refractorycancers has also been observed as part of this study.

A total of 68 patients have been entered into this dose escalationstudy, of which 49 were evaluable for clinical response in that theyhave received at least 2 Cycles of NUC-1031 and were therefore eligiblefor a RECIST 1.1 assessment.

TABLE 1 Best Overall Response in the ProGem1 Study Patients n = 68 BestOverall Response (to date) Evaluable n = 49 Partial Responses  5 (10%)Stable Disease 33 (67%) Progressive Disease 11 (22%) Non Evaluable n =19NUC-1031 was administered as a 5 to 30 minute intravenous slow bolusinjection.Schedule A: NUC-1031 was administered on days 1, 8, 15 of a 4 weeklyCycle.Schedule B: NUC-1031 was administered on days 1, 5, 8, 12, 15, 19, of a4 weekly Cycle.Evaluable Patients [n=49]

Evaluable patients were patients that received ≥2 Cycles of NUC-1031 andwere therefore eligible for a RECIST 1.1 assessment at the end of Cycle2. Where the disease response duration, measured in months, is followedby a “+” this indicates ongoing disease control as of the latest cut-offdate.

Patient 004 Breast Cancer: Stable Disease

Female (67 years)

Diagnosed with Grade 2 invasive ductal carcinoma of the breast (ER+ve,HER2−ve) in 2002. As first line treatment she was given surgery andreceived adjuvant epirubicin+docetaxel, radiotherapy and maintenancehormone therapy with tamoxifen, then anastrozole until 2010. In 2010,patient was diagnosed with metastatic disease in the bone and wastreated with palliative radiotherapy and commenced on ibondronate andfulvestrant as second line treatment.

Disease progression was noted in 2012 and she was given third linechemotherapy with capecitabine and navelbine for 4 months, but while ontreatment her disease progressed, with new liver and lung metastases.The patient was commenced on a PI3K inhibitor (Phase 1 study) inSeptember 2012 and received 2 Cycles (2 months), but disease progressed,with increase in the size of liver metastases, while on treatment.

Commenced NUC-1031 on 3 Dec. 2012 on 500 mg/m² weekly. Completed 6Cycles and tolerated treatment well. Patient had Stable Disease at endof study and requested compassionate continuation of a 7^(th) Cycle ofNUC-1031, then elected for a ‘drug holiday’ after Cycle 7. Remainedstable for further 5 months with no further treatment before diseaseprogression.

Stable Disease to RECIST (12 months).

Patient 005 Ovarian Cancer: Stable Disease

Female (58 years)

Diagnosed with Stage 3c (Grade 3) bilateral serous ovarian cancer in2009. On the 23 Jun. 2009 the patient had a total abdominal hysterectomyand salpingo-oopherectomy performed, but unresectable omental depositswere left in situ.

As first line chemotherapy, in October 2009 the patient received 6Cycles of carboplatin+paclitaxel, but developed an allergic reaction tocarboplatin at final treatment Cycle.

First relapse was 8 months later and in December 2010 patient commencedon 6 Cycles of Caelyx plus VEGFR-2 inhibitor (Phase II clinical study),remaining on VEGFR-2 monotherapy as maintenance. Second relapse was 9months later, with a rise in CA125 and CT evidence of a left sidedpelvic mass measuring 3.2 cm. Commenced on third line chemotherapy andreceived 6 Cycles of weekly paclitaxel and there was an initial moderateresponse with a fall in CA125 and some reduction in tumour volume.Disease progression was confirmed 5 months later with rising CA125levels and increased tumour size. Patient received the last dose ofpaclitaxel in March 2012.

Commenced NUC-1031 on 7 Jan. 2013 on 500 mg/m² weekly. Completed 6Cycles as per Study protocol, with Stable Disease, and then a further 6months of NUC-1031, resulting in 12 months of treatment. Patienttolerated treatment well. CA125 levels dropped from 208 at start ofstudy to 140 at the end of Cycle 6. Patient elected to stop therapyafter 12 months, and relapsed 3 months after stopping treatment.

Stable Disease to RECIST (15 months).

Patient 006 Cholangiocarcinoma: Stable Disease

Male (43 years)

Diagnosed with a primary cholangiocarcinoma in 2009. A Whipple procedurewas performed and the patient was given 6 cycles of adjuvantgemcitabine. On disease recurrence in February 2012, the patient wascommenced on CapeOx until July 2012. Later that year a CT scan showedbone metastases and these were treated with a course of radiotherapy tothe lower back.

Commenced NUC-1031 on 31 Jan. 2013 on 375 mg/m² twice weekly, andcompleted 2 Cycles. Changed to schedule A at 500 mg/m² and received 1further dose. Reduction in CA19.9 from 125,002 to 59,285 after only twodoses of NUC-1031. CT scan revealed a reduction in metastatic lunglesion and lymph node from baseline.

Stable Disease to RECIST (3 months).

Patient 007 Colorectal Cancer: Stable Disease

Male (73 years)

Diagnosed with colorectal cancer in 2008. Following 6 Cycles of FOLFOXhad the primary tumour removed in April 2009. Further surgery in August2009 when a right hepatectomy and ileostomy were performed. In July 2010was included in the PICCOLO trial (panitumumab+irinotecan). Six monthslater received radiofrequency ablation (RFA) plus biliary stenting foradditional complications. In December 2011 he received 7 Cycles ofcetuximab+irinotecan+5-FU, but with subsequent disease progression.

Commenced NUC-1031 on 11 Feb. 2013 on 375 mg/m² twice weekly, andcompleted 0.5 of a Cycle.

Following treatment delays, and at the patient's request forconvenience, he was changed to a weekly schedule at 500 mg/m² andcompleted 2 Cycles. Over the treatment period he developedthrombocytopaenia (G3) (possible linked to splenomegaly seen atbaseline). An ultrasound scan on 25 Feb. 2013 showed compression ofportal vein, with possible thrombosis, as a result of the splenomegaly.Reduction in CEA from 361 to 286 and CA19.9 from 3,151 to 2,957.

Stable Disease to RECIST (3 months).

Patient 008 Cancer of Unknown Primary: Stable Disease

Female (37 years)

Diagnosed with a retroperitoneal mass and metastatic disease of unknownprimary in 2012. Rapid tumour progression following 4 Cycles ofgemcitabine+cisplatin from August 2012.

Commenced NUC-1031 on 12 Feb. 2013 on 375 mg/m² twice weekly, andcompleted 2 Cycles. During Cycle 1 patient had symptomatic relief ontreatment and a general improvement in mood and wellbeing. Developedtransient (G3) transaminitis (ALT and AST). Lymphoedema, which waspresent at baseline, was progressing during Cycle 2.

Also developed a pleural effusion (G3) that was assessed as unlikely tobe related to study drug.Stable Disease to RECIST (3 months).

Patient 010 Endometrial Cancer: Stable Disease

Female (60 years)

Diagnosed with endometrial cancer (stage IV, grade 3) in 2012. InFebruary 2012 the patient received radiotherapy to a pelvic mass and 6Cycles of carboplatin+paclitaxel. On disease progression in November2012 received 3 Cycles of paclitaxel, and then rapidly progressedthrough a Cycle of Megace in January 2013.

Commenced NUC-1031 on 19 Mar. 2013 on 750 mg/m² weekly, and completed 1Cycle. Developed neutropenia (G3) during Cycle 1, which resolved after afew days, following intervention with GCSF. Dose reduced to 500 mg/m²for Cycle 2 and completed 5 Cycles at this dose. Patient had transientGrade 3 anaemia. Reduction in CA125 from 727 to 488. Patient had StableDisease on study and this was maintained for a further 3 months afterstopping NUC-1031.

Stable Disease to RECIST (9 months).

Patient 011 Uterine Carcinosarcoma: Stable Disease

Female (67 years)

Diagnosed with uterine carcinosarcoma (recurrent MMMT of the uterus) andliver, lung and para-aortic nodal metastases. Surgery was performed inJune 2011 with a radical hysterectomy and bilateralsalpingo-oophorectomy. From June 2011 to November 2012 the patientreceived adjuvant cisplatin and doxorubicin-6 cycles completed (goodresponse to treatment with almost complete remission).

June 2012 underwent further surgery with an anterior exenteration,dissection of rectum and repair and formation of ileal conduit withend-to-end anastomosis. Further surgery in June 2012 when she underwenta laparotomy with implantation of a ureter in to her ileal conduit and aHartman's procedure for a rectal fistula. March 2013 completed 6 Cyclesof weekly paclitaxel but with tumour progression through this course oftreatment.

Commenced NUC-1031 on 15 Apr. 2013 on 375 mg/m² twice weekly, andcompleted 2.6 Cycles. Patient had stable disease to RECIST with areduction in tumour volume of 26%. During treatment had transientneutropaenia (G3) and a low haemoglobin (G2). At patient's request,changed to weekly schedule at 625 mg/m² for Cycle 4, and completed 1further Cycle. The end of Cycle 4 (end Month 4) CT scan in August 2013showed progressive disease. Three new lesions appeared: 2 in the lung,and 1 retro cava lymph node. The original lesion in the liver hadcontinued to decrease in size and the other target lesion in the lunghad remained stable. The patient was withdrawn from the study.

Stable Disease to RECIST (4 months).

Patient 012 Cholangiocarcinoma: Stable Disease

Female (48 years)

Diagnosed with Stage IV, Grade 3 cholangiocarcinoma in 2013 followinginvestigation for right-sided abdominal pain. CT scan showed liver, lungand peritoneal metastases. From January-Aprril 2013 the patient received3 Cycles of cisplatin+gemcitabine, with rapid disease progression.

Commenced NUC-1031 on 16 May 2013 on 375 mg/m² twice weekly, andcompleted 3 Cycles. At patient's request changed to weekly schedule at625 mg/m² for Cycle 4, completed 3 further Cycles and toleratedtreatment well. Patient had Stable Disease at end of study and had afurther two Cycles, with a total of 8 Cycles.

Stable Disease to RECIST (8 months).

Patient 013 Cervical Cancer: Partial Response

Female (51 years)

Diagnosed with inoperable, poorly differentiated squamous cell cervicalcancer (Stage 2b, G2/3) in September 2011. She was treated withcisplatin (4 Cycles) plus radiotherapy and was said by the referringclinician to have a “good response”.

In July 2012 the patient developed disease progression and, between July2012 and November 2012, was given 6 doses of carboplatin+paclitaxel plusCediranib (CIRCCA trail) and achieved stable disease. In April 2013 MRIscan showed disease progression with increased iliac lymph nodeinvolvement.

Commenced on NUC-1031 on 28 May 2013 on 750 mg/m² weekly, and completed2 Cycles. Dose reduced to 625 mg/m² and completed 4 further Cycles. Hadlower pelvic pain prior to study but had relief of this pain ontreatment, with significant reduction of opioid usage. The patient hadproblems with recurrent urinary tract infections both before startingNUC-1031 and during treatment because of bilateral metal uretericstents.

Patient had a Partial Response at completion of the ProGem1 study andthen completed a further 3 Cycles on a reduced dose of 500 mg/m² weekly,with a total of 9 Cycles of NUC-1031. Patient's tumour has shrunk to theextent that she was being re-evaluated for further debulking surgery.

Partial Response to RECIST (9 months, PFS 11 months).

Patient 014 Mesothelioma: Progressive Disease

Female (51 years)

Patient diagnosed with a recurrent epitheliod mesothelioma in the righthemi-thorax in 2012. Received 4 Cycles of permetrexed+carboplatin. Onprogression in December 2012 entered a clinical study to receivedasatinib but was unresponsive and had disease progression.

Commenced on NUC-1031 on 20 Jun. 2013 on 750 mg/m² weekly, and completed1 Cycle. Dose reduced to 625 mg/m² and completed 1 further Cycle.Withdrawn from study. Progressive Disease.

Patient 015 Cancer (Unknown Primary): Partial Response

Male (54 years)

Diagnosed with cancer of unknown primary in September 2012, having beeninvestigated for symptoms of abdominal pain.

He was noted at that time to have liver and lung metastases. A liverbiopsy showed a poorly differentiated carcinoma with focal glandulardifferentiation. Received 8 Cycles of epirubicin+cisplatin+capecitabinefrom October 2012 until April 2013 within the “CUP” clinical study butdeveloped Progressive Disease with oedema, pleural effusion and ascites,with a 20 kg weight gain. Patient experienced severe nausea and vomitingand fatigue on this regimen.

Commenced on NUC-1031 on 20 Jun. 2013 on 750 mg/m² weekly, and received2 doses. Dose reduced to 625 mg/m² to complete Cycle 1 and received 5further Cycles at the lower dose. On study entry patient had markedlower limb oedema and abdominal ascites (approximately 20 Kg). FollowingCycle 1 it was reported that the oedema and ascites had gone and thepatient was feeling much better. During Cycle 1 Day 8 developedthrombocytopenia (G3), lymphopenia (G3) and neutropenia (G2) whichcaused a two week treatment delay. End of Cycle 2 scan showed areduction in all target lesions with a RECIST assessment of StableDisease. End of Cycle 4 scan showed further reduction in all targetlesions, with a RECIST assessment of a Partial Response, which wassustained until end of study. Requested compassionate continuation andcompleted 3 further Cycles at this dose. Was beginning to show aconsistent drop in blood counts following Day 8 of each Cycle.

From Cycle 10 dose reduced further to 500 mg/m² with the desired effectand completed a further 10 Cycles (19 in total).

Most recent CT scan on 9 Jan. 2015 showed sustained Partial Response(approximately 58% reduction in tumour size) and the target mesentericnode no longer visible. Had fluid build up in both legs during Cycle 10but responded well to spironolactone and completely resolved. ReplacedHickman line on 12 Dec. 2014 following 19 months in situ, with noadverse effect. Patient remains clinically very well. Followingdiscussions with CI Patient is happy to have a treatment break and willbe referred back to oncologist to discuss options.

Partial Response to RECIST (20+ months; PFS 24+ months) ongoing.

Patient 017 Lung Cancer: Partial Response

Female (60 years)

Diagnosed with lung adenocarcinoma, with lung, liver and adrenalmetastases in September 2011. Also had disease in mediastinal, intraabdominal and cervical lymph nodes. The patient had a pleurodesisperformed in December 2011 followed by 3 Cycles of cisplatin+pemetrexedwhich she completed in March 2012 and achieved a partial response. FromMarch-September 2012 she received a total of 6 Cycles of docetaxel, witha partial response. In April 2013 she had disease progression and wasre-challenged with docetaxel, but progressed through 2 Cycles ofdocetaxel.

Commenced on NUC-1031 in July 2013 on 750 mg/m² weekly and received 1dose. Dose reduced to 625 mg/m² and she received 2 doses to completeCycle 1 and has completed 5 further Cycles. Had 4 weeks of treatmentdelays due to lung infections and low platelets. Significant response indiseased lymph nodes, particularly in the neck.

Target lesions continued to shrink which was evident on post Cycle 2 andCycle 4 CT scans. At this stage, RECIST assessment classified theresponse as Stable Disease.

At the end of Cycle 6 a CT scan showed further reduction and the RECISTassessment was changed to a Partial Response in all target lesions atend of study. Requested continuation on a compassionate use basis andcompleted 3 further Cycles. Patient tolerated NUC-1031 well, withimprovement in hoarse voice and dysphagia. End of Cycle 9 scan on 17Apr. 2014 showed Progressive Disease with a growth on target lesions andnew hepatic lesions. Withdrawn from study.

Partial Response to RECIST (3 months; PFS 10 months).

Patient 018 Lung Cancer: Stable Disease

Female (65 years)

Diagnosed with squamous cell lung cancer May 2011. Received

gemcitabine+cisplatin, 6 Cycles from June to October 2011 within theSQUIRE trial. Disease relapse in April 2012 and she received singlefraction palliative radiotherapy to right hilum and docetaxel 6 Cyclesfrom May until September 2012.

Progressive disease October 2012. Commenced erlotinib December 2012 for3 months but in March 2013 was found to have progressive disease in theright hilum.

Commenced on NUC-1031 on 25 Jul. 2013 on 625 mg/m² weekly, and completed4 Cycles. Stable disease to RECIST (4 months). Patient withdrawn fromstudy due to symptomatic deterioration caused by vena caval obstruction.Received low dose radiotherapy in an attempt to resolve the obstructionand recommenced NUC-1031 on the compassionate access programme.Following one further dose of NUC-1031 it was agreed to withdraw patientfrom the compassionate access programme. The bulk of the patient'sdisease was stable on withdrawal.

Stable Disease to RECIST (4 months).

Patient 021 Fallopian Tube Cancer: Partial Response

Female (61 years)

Diagnosed with recurrent Stage 2a, Grade 2 endometrioid adenocarcinomaof the ovary in 2008. She received 6 Cycles of carboplatin+paclitaxel,completed October 2008. In June 2011 she relapsed and was noted to havepleural, subcapsular liver, omental and mesenteric tumour nodules andwas recruited into the ICON6 study, receiving 6 Cycles of carboplatinplus paclitaxel+/−cediranib. She achieved a partial response to therapy.Remained on maintenance cediranib until March 2012 when treatment wasdiscontinued due to rising CA125 and progressive peritoneal disease.

From March to July 2012 she received 6 Cycles of weekly paclitaxel withgood radiological response initially of the peritoneal disease.

In February 2013 she was found to have a new effusion and an increase inthe peritoneal disease. She commenced carboplatin+paclitaxel and dailyAKT inhibitor on the AKTRES study but with disease progression (newpelvic mass) in May 2013 after 3 Cycles.

Commenced on NUC-1031 on 28 Aug. 2013 on 625 mg/m² weekly, and completed6 Cycles. Noticed a significant reduction in abdominal ascites; requireddrainage every two weeks prior to coming on study and has not requiredfurther drainage since commencing NUC-1031. Patient tolerated NUC-1031well. Stable disease to RECIST at end of study. Requested continuationon compassionate use basis and completed one further Cycle. End of Cycle7 CT scan on the 26 Feb. 2014 showed a further reduction in tumourvolume which confirmed Partial Response to RECIST. Significant CA125Response: 91% reduction from baseline (372) to end of Cycle 6 (35).

Best overall response to date is Partial Response (3 months) accordingto RECIST or Partial Response (9 months) according to GCIG criteria.

Partial Response. Patient 024 Cancer of Unknown Primary: ProgressiveDisease

Female (51 years)

Diagnosed with cancer of unknown primary in April 2012. Received CAPOX,8 Cycles from April to October 2012. Received irinotecan in October 2012with addition of bevacizumab in November 2012, but without response.

Commenced on NUC-1031 on 26 Sep. 2013 on 675 mg/m² weekly, and completed2 Cycles. End of Cycle 2 CT scan showed progressive disease.

Progressive Disease. Patient 025 Mesothelioma: Stable Disease

Male (54 years)

Diagnosed with T4 N3 M0 epithelioid mesothelioma of the right lung inMarch 2013. Received 4 Cycles of pemetrexed+cisplatin from May to August2013.

Commenced on NUC-1031 on 23 Oct. 2013 on 725 mg/m² weekly, and completed4 Cycles. End of C4 CT Scan showed Progressive Disease. Withdrawn fromstudy.

Stable Disease to RECIST (4 months).

Patient 026 Colorectal Cancer: Stable Disease

Female (63 years)

Diagnosed with colorectal cancer, T4 N2, with lung and bladdermetastases in February 2007. Received adjuvant FOLFOX, 12 Cycles,November 2007. Developed pelvic recurrent disease and receivedcapecitabine 2009.

On relapse in 2012 received FOLFIRI in September 2012 andcapecitabine+irinotecan until January 2013. In July 2013 CT showedProgressive Disease, presacral tumour recurrence causing destruction ofsacrum, and a lung nodule.

Commenced on NUC-1031 on 17 Oct. 2013 on 725 mg/m² weekly, and completed4 Cycles. Significant improvement in pain, with dramatic reduction inuse of opioid analgesia. End of Cycle 4 CT Scan showed ProgressiveDisease.

Stable Disease to RECIST (4 months).

Patient 027 Ovarian Cancer: Stable Disease

Female (46 years)

Diagnosed with serous adenocarcinoma of both ovaries in December 2009.Following total hysterectomy, bilateral salpingo-oophorectomy andomentectomy she received 6 Cycles carboplatin+paclitaxel and achieved aComplete Response in May 2010. The patient relapsed in June 2011 andreceived carboplatin+paclitaxel 6 Cycles (ICON6 Study). In December 2012the patient was given a further 3 Cycles of gemcitabine+carboplatin buthad an allergic reaction to carboplatin which was switched to cisplatin.She completed 6 Cycles in total and achieved a partial response in April2013. This was followed by 6 months of tamoxifen but in July 2013 a CTscan showed new mediastinal lymph node involvement and the CA125 levelsincreased. A CT scan in October 2013 showed an increase in the size ofperitoneal deposits.

Commenced on NUC-1031 on 30 Oct. 2013 on 725 mg/m² weekly. Developedelevated ALT (G3) following Cycle 1 Day 1, raised from 96 at baseline to256 on day 7, a DLT for this cohort. ALT recovered to G2 a few dayslater to allow patient to receive Cycle 1 Day 8 at the reduced dose of675 mg/m². Completed Cycle 1 at reduced dose and went on to receive afurther 3 Cycles. Patient achieved Stable Disease to RECIST with areduction in tumour volume of 23%. CA125 has reduced from 188 atbaseline to 99 at end of Cycle 6. Dose was further reduced for Cycle 5to 625 mg/m² due to mild neutropenia. Completed study at this dose withno further issues. Requested compassionate continuation and received 1further Cycle.

Stable Disease to RECIST (8 months).

Patient 029 Breast Cancer: Stable Disease

Female (53 years)

Diagnosed with metastatic breast cancer (ER and PGR positive), withmultiple bone and hepatic metastases in 2002. Received 6 Cycles of FEC,adjuvant radiotherapy and tamoxifen with goserelin. In 2010 new bonemetastases detected and treated with Zoladex, letrozole and pamidronate.July 2011, switched to Zoladex and exemestane, which was augmented withFaslodex in November. On further progression in 2012 receivedcapecitabine+Zometa, followed by paclitaxel for 3 Cycles only. Commencedtreatment with rucaparib in May 2013. Progressive hepatic disease inJuly 2013 and received gemcitabine+carboplatin for 3 Cycles.

Commenced on NUC-1031 on 14 Nov. 2013 on 725 mg/m² weekly. Completed 3Cycles. Unfortunately suffered a fatal cardiac arrest while at home, notstudy related.

Stable Disease to RECIST (4 months).

Patient 030 Ovarian Cancer: Stable Disease

Female (62 years)

Diagnosed with serous adenocarcinoma of the ovary in 2012. Receivedadjuvant carboplatin+paclitaxel for 6 Cycles to July 2012, achievedcomplete response. Progressive disease in August 2013, commencedcarboplatin+caelyx, progressed following 3 Cycles.

Commenced on NUC-1031 on 21 Nov. 2013 on 725 mg/m² weekly. Completed 3Cycles and tolerated study drug well. End of Cycle 2 CT scan showedStable Disease to RECIST. Unstable dietary issues resulting indehydration and malnutrition, which led to lengthy treatment delays.Withdrawn from study.

Stable Disease to RECIST (3 months).

Patient 031 Cholangiocarcinoma: Progressive Disease

Female (76 years)

Diagnosed with cholangiocarcinoma in July 2013. On the 27^(th) July sheunderwent a modified Whipple's procedure. At the time of surgery she wasnoted to have multiple liver metastases. In August 2013 she commencedgemcitabine+oxaliplatin which was given every two weeks for 6 Cycles.

Commenced on NUC-1031 on 9 Dec. 2013 on 750 mg/m² weekly and completed 2Cycles. End of Cycle 2 CT scan showed Progressive Disease.

Withdrawn from study.

Patient 032 Oesophageal Cancer: Stable Disease

Male (56 years)

Diagnosed with squamous cell carcinoma of the oesophagus in June 2013.Received 3 Cycles of cisplatin+capecitabine from July to September 2013.Progressive disease with peritoneal and lung metastases. Oesophagealstent inserted in October 2013 to control symptoms of dysphagia.

Commenced on NUC-1031 on 16 Dec. 2013 on 750 mg/m² weekly and completed2 Cycles. End of Cycle 2 CT scan showed Stable Disease to RECIST.Patient was having difficulties with a lung/trachea fistulae. Withdrawnfrom study due to clinical progression.

Stable Disease to RECIST (2 months).

Patient 033 Cholangiocarcinoma: Stable Disease

Female (37 years)

Diagnosed with advanced cholangiocarcinoma in June 2013 with liver,peritoneal and para aortic lymph node metastases and small pulmonarynodules. In June she had a liver biopsy which showed a probable poorlydifferentiated cholangiocarcinoma, with some features to suggest a liverprimary. In July 2013 she commenced chemotherapy with gemcitabine andcisplatin and received 6 Cycles omitting some of Cycle 5 due to anadmission for neutropenic sepsis.

Unfortunately, although her interval scan showed a partial response herpost treatment CT scan on the 28 Nov. 2013 showed progressive diseasewith a stable liver lesion but an increase in the size of her pulmonarymetastases and some new peritoneal deposits. She also is known to have alytic sternal lesion.

Commenced on NUC-1031 on 3 Jan. 2014 on 750 mg/m² weekly and completed 4Cycles. End of Cycle 2 CT scan showed Stable Disease to RECIST. End ofCycle 4 scan showed progressive disease and patient was withdrawn fromthe study.

Stable Disease to RECIST (3 months)

Patient 036 Renal Carcinoma: Stable Disease

Male (20 years)

Diagnosed with medullary cell renal carcinoma in December 2012. Received5 Cycles of gemcitabine+paclitaxel+carboplatin from January until July2013 resulting in Stable Disease during treatment. Patient had treatmentdelays due to thrombocytopenia and neutropenia which requiredintervention with G-CSF. Following relapse in July 2013 commencedgemcitabine+doxorubicin but progressed through 2 Cycles in September2013.

Commenced on NUC-1031 28 Jan. 2014 on 825 mg/m² weekly and completed 4Cycles. Patient experienced fatigue and tiredness during Cycle 1.Lorazepam was discontinued and he became much more alert. He reportedthat he was tolerating NUC-1031 much better than his previous regimen.End of Cycle 4 CT scan showed sustained Stable Disease with a reductionin tumour volume of 7%. Following Cycle 5 Day 1 developedthrombocytopenia (G3) and dose was reduced to 750 mg/m². Following Cycle5 Day 8 developed fatigue, loss of appetite and did not present for anyfurther treatment. Withdrawn from study due to clinical progression.

Stable Disease to RECIST (5 months).

Patient 037 Pancreatic Cancer: Partial Response

Female (70 years)

Diagnosed with pancreatic adenocarcinoma in March 2013. Whipple'sprocedure was planned for 26 Mar. 2013 but due to extensive adhesionsthe cancer was non-resectable, but biopsies were taken. Liver wedgeresection confirmed metastatic disease. Histology showed moderatelydifferentiated adenocarcinoma. Patient received 6 Cycles of gemcitabinefrom May to October 2013. CT scan November 2013 suggested partialresponse of pancreatic tumour, but with new metastases in the lateralleft lobe of the liver.

Commenced on NUC-1031 4 Feb. 2014 on 1,000 mg/m² weekly and received 1Cycle at this dose. At this time the DSMC decided to reduce the dose inall patients in this cohort to 900 mg/m² due to a DLT in one patient(patient 039). Patient received one further Cycle at the new dose. Endof Cycle 2 CT scan showed Stable Disease to RECIST with an 18.4%reduction in tumour volume.

Pain in abdomen and back had significantly improved; patient was onoxycontin 80 mg bd and had now stopped all morphine. Also had a verysignificant drop in tumour markers: CA19.9 from 15,000 at baseline to4,000 and CEA from 536 at baseline to 42. Fatigue had become a majorissue following each cycle. CT scan on 29 Apr. 2014 showed a furtherreduction in tumour volume to 30% to achieve a Partial Response toRECIST. Patient had loss of appetite, severe fatigue and was withdrawnfrom the study.

Partial Response to RECIST (1 month; PFS 4 months).

Patient 038 Ovarian Cancer: Progressive Disease

Female (65 years)

Diagnosed with recurrent stage 3c grade 3 serous adenocarcinoma of theovary in 2000. She underwent a total abdominal hysterectomy, withbilateral salpingo-oophorectomy and debulking surgery, leaving minimalresidual disease, in November 2000. Received 6 Cycles of 3 weeklycarboplatin+paclitaxel until March 2001. Following relapse in 2002received 6 cycles of Carboplatin plus Etoposide to complete remission inMarch 2003. Further relapse in 2005 and had more debulking surgery,followed by carboplatin+gemcitabine×6 Cycles which finished in July 2006with complete response. Additional debulking surgery, includingsplenectomy, was required following relapse in 2009. This was followedby 6 Cycles of carboplatin+caelyx and a complete response was achieved.A right pelvic recurrence near the right external iliac vessel, whichwas considered to be inoperable, was noted in 2010. The patient receivedcarboplatin and paclitaxel for 6 Cycles, which she completed in April2011, with a partial response. In October 2011 she showed evidence ofprogression, received topotecan for 6 Cycles till March 2012 and hadstable disease. At this time she underwent insertion of a right uretericstent for hydronephrosis. In June 2012, after further diseaseprogression with new bilateral lung metastases, she was started onweekly carboplatin+paclitaxol+bevacizumab to March 2013 followed bymaintenance bevacizumab+letrozole to September 2013. This was followedby 3 Cycles of cyclophosphamide+bevacizumab, but interval scan showedprogressive disease and on 11 Nov. 2013 her treatment was discontinued.

Right ureteric stent changed January 2014.

Commenced on NUC-1031 on 4 Mar. 2014 on 900 mg/m² weekly and completed 2Cycles. Main toxicity was delayed onset fatigue, which set in on days 3to 4. End of Cycle 2 scan showed progressive disease with a 25% increasein tumour volume. Withdrawn from the study.

Progressive Disease. Patient 040 Cholangiocarcinoma: Stable Disease

Female (69 years)

Diagnosed in May 2013 with intrahepatic grade 2 cholangiocarcinoma, with11 cm liver mass obstructing the common bile duct and causing jaundice.A biliary stent was inserted. Received 7 Cycles Gemcitabine+Cisplatinfrom August to December.

Commenced on NUC-1031 on 20 Feb. 2014 on 1,000 mg/m² weekly and received1 dose. Presented for Cycle 1 Day 8 on 27^(th) February with fever,rigors and an elevated bilirubin. Was admitted, and source of infection(G3) was a stent blocked with tumour and a biliary tract cyst. Two newstents were working well. Cycle 1 was completed at 900 mg/m² due to DLTin that cohort. Completed 3 Cycles. End of Cycle 2 CT scan on 23^(rd)May showed Stable Disease to RECIST with slight reduction in tumourvolume from 85 at base to 82.1. CA 19.9 dropped from 664 at baseline to155 on 4^(th) June. Was admitted with delirium in June 2014 anddiagnosed with a urinary tract infection. Further investigation alsorevealed progressive disease in the liver. Withdrawn from study.

Stable Disease to RECIST (4 months).

Patient 041 Breast Cancer: Stable Disease

Female (54 years)

Diagnosed with metastatic invasive ductal breast cancer (ER and PR+ve)with bilateral axillary nodes, lung and liver metastases. Received FEC×3Cycles, paclitaxel×9 Cycles, capecitabine×8 Cycles, euribulin×3 Cyclesand gemcitabine+carboplatin×1 dose. Her last dose of chemotherapy was onthe 19 Dec. 2013 and the last CT scan before enrolment showedprogressive disease.

Commenced on NUC-1031 on 18 Mar. 2014 on 900 mg/m² weekly and completed3 Cycles. Had 2 treatment delays following Cycle 1 Day 8 and Day 15 dueto neutropenia, which resolved spontaneously within one week. End ofCycle 2 CT scan showed Stable Disease to RECIST. CA 15.3 tumour markerwas 726 at base 845 at C2 and was 824 on 19 May 2014. (This tumourmarker has always been a reliable indicator of response in the past).Experiencing fatigue (G3) during Cycle 3 but had managed to reduceopioids significantly. NUC-1031 dose reduced to 825 mg/m² for Cycle 4.End of Cycle 4 scan showed progressive disease with increase at targetsites and new bone lesions. Withdrawn from study.Stable Disease to RECIST (4 months)

Patient 042 Cholangiocarcinoma: Progressive Disease

Male (48 years)

Diagnosed with metastatic cholangiocarcinoma in January 2013. Partialhepatectomy in February 2013. Commenced on the BILCAP trial observationarm (comparing capecitabine with observation after surgery for biliarytract cancer). On progression commenced on gemcitabine and cisplatin×6Cycles. On further progression in November 2013 he commencedcapecitabine. However, this was stopped after two months as he developedangina. On further progression he was commenced on 5FU×6 weeks but hadprogressive disease with lung, liver and bone metastases.

Last treatment was in January 2014. Received palliative radiotherapy forpain in right shoulder bone metastasis on the 20 Jan. 2014.

Commenced on NUC-1031 on 18 Mar. 2014 on 900 mg/m² weekly and completed2 Cycles. Experienced delayed onset fatigue (G2) on days 3-5 followingstudy drug. End of Cycle 2 CT scan showed a 9% reduction in tumourvolume of primary target lesion but showed new pulmonary and bonelesions. Withdrawn from the study. Progressive Disease.

Patient 043 Ovarian Cancer: Stable Disease

Female (54 years)

Diagnosed with stage 4, Grade 3 papillary serous peritoneal cancer. Atotal abdominal hysterectomy, with bilateral salpingo-oophorectomy andomentectomy performed in September 2007. Receivedcarboplatin+paclitaxel×4 Cycles followed by 2 Cycles of Carboplatinalone, due to neuropathy, and completed course in January 2008. In April2011 underwent secondary debulking surgery for recurrent pelvic mass.Patient did not wish to have adjuvant chemotherapy or radiotherapy.Further recurrence of disease July 2012 and a stent inserted forhydronephrosis. Recurrent disease in August 2012 and commencedcarboplatin+gemcitabine. CT scan in March 2013 revealed diseaseprogression; patient completed 6×Cycles of Caelyx in October 2013.Progressive disease early 2014 with pleural effusion, which requiredvery regular drainage.

Commenced on NUC-1031 on 20 Mar. 2014 on 900 mg/m² weekly and completed6 Cycles. Received PET scan on 7 Apr. 2014 which showed stable diseaseand SUV had gone down in some target tumours. CA125 had reduced from1.099 at baseline to 783 at the beginning of Cycle 2. The volume offluid from the pleural effusion also reducing (was draining 300 ml perweek, now 150 ml per week). Developed delayed onset fatigue (G2) on days4 and 5 and G1 at all other times. End of Cycle 6 CT scan showed StableDisease to RECIST with an overall 10% reduction in tumour volume frombaseline. CA125 fell from 1,099 at baseline to 910 on 15^(th) July.Completed study and requested compassionate continuation. Dose reducedto 750 mg/m² for Cycle 7 and received 1 further Cycle. Leg oedemadeveloped to G3, no disease progression but new treatment options soughtand withdrawn from study.

Stable Disease to RECIST (13 months).

Patient 044 Lung Cancer: Stable Disease

Female (64 years)

Diagnosed with adenocarcinoma of the lung (left lower lobe) February2010 following unresolved cough for 7 months. Between January 2011-April2012 patient received Gefitinib 250 m, but had progressive disease.Received Afatinib from April 2012 to November 2012 (but dose reductiondue to skin toxicity) but again with progressive disease. From November2012 to June 2013 patient given Erlotinib after worsening cough andprogression of the primary lesion. In June 2013 developed abnormalvision and black shadows in left eye and found to have choroidalmetastases for which she received radiotherapy to both eyes, withimprovement of her vision. In June 2013—started pemetrexed+carboplatinfor 6 Cycles followed by maintenance pemetrexed until 6 Feb. 2014 whenprogressive disease was diagnosed.

Commenced on NUC-1031 on 27 Mar. 2014 at 900 mg/m² weekly and hadcompleted 2 Cycles. Had a G3 lung infection on 19^(th) April whichresponded well to antibiotics. Commenced Cycle 2 on a reduced dose, 825mg/m² and completed a further 2 Cycles. End of Cycle 2 CT scan showedStable Disease to RECIST with a 10% reduction in tumour volume. Had 2treatment delays for ascites drainage and two more for thrombocytopenia.End of Cycle 4 scan showed progressive disease in lung and new livermetastases. Withdrawn from study.

Stable Disease to RECIST (5 months).

Patient 046 Adrenal Carcinoma: Stable Disease

Male (36 years)

Diagnosed in August 2011 with a large 20×19×9 cm adrenocorticalcarcinoma. Ki 67 35-40% mitotic count 25/50hpf, Weiss score 6 withtumour extending to 0.3 mm of external margins but no renal involvement.Received adjuvant mitotane until January 2012 when it was stopped due tonausea and diarrhoea. Recommenced mitotane in June 2012. Relapsed inJune 2013 with new liver metastases and commenced etoposide+carboplatin.

Staging CT scan on September 2013 showed differential response butoverall stable disease, and he received a further 3 Cycles and remainedstable on completion in November 2013. In January 2014 showedprogressive disease in the liver.

Commenced on NUC-1031 on 16 Apr. 2014 on 1,000 mg/m² weekly andcompleted 1 Cycle. During Cycle 1 experienced delayed onset fatigue,nausea and vomiting (all G3). Dose was reduced to 900 mg/m² for Cycle 2and received a further 5 Cycles on this dose to complete the study. Endof Cycle 6 CT scan showed a 12.6% reduction in tumour volume frombaseline. Requested compassionate continuation and completed 3 furtherCycles. Developed fatigue (G3) and neutropenia (G2) following Cycle 8D1, was dose reduced to 750 mg/m² for Cycle 8 and received 2 furtherCycles. Though improved to G1, fatigue continued, also developed nauseaand vomiting and dose was further reduced to 625 mg/m2 for C11. Received2 further doses. Withdrawn from study.

Stable Disease to RECIST (11 months).

Patient 048 Ovarian Cancer: Stable Disease

Female (63 years)

Diagnosed with stage 1a granulosa cell tumour of the ovary in 2000 andhad total abdominal hysterectomy and bilateralsalpingo-oophorectomy+omentectomy. On recurrence in February 2004commenced 3 cycles of BEP (bleomycin+etoposide+cisplatin) to diseaseprogression. Secondary debulking in July 2004.

In June 2006 underwent partial hepatectomy, splenectomy and excision ofdeposits from stomach and peritoneam. This was followed byradiofrequency ablation to liver deposits. Underwent laparotomy andresection of 4 further metastatic deposits in September 2008. In March2009 commenced 3 weekly carboplatin+paclitaxel, with a mixed response.Changed to weekly carboplatin+low dose paclitaxel on May 12, 2009.Completed in October 2009 with PR and CA-125 negative. Further debulkingsurgery October 2011, with complications requiring prolonged stay inITU. CT scan in April 2014 showed lesion in segment 8 of liver hadincreased in size, new small peritoneal metastases in the gastro-hepaticligament and some new small volume lymphadenopathy in the small bowelmesentery and further small peritoneal deposits in the pelvis around therecto-sigmoid junction.

Commenced on NUC-1031 on 29 Apr. 2014 on 1,000 mg/m weekly and received1 dose. Following Cycle 1 Day 1 developed; ALT, (G3, a DLT); AST andneutropaenia (G2); ALP (G1). Dose reduced to 900 mg/m² for Cycle 1 Day8. ALT returned to G3 following days 8 and 15. Dose for Cycle 2 reducedto 825 mg/m². Results from PET scan at end of Cycle 1 showed stabledisease and reduction in SUV at target sites. In January 2014 inhibin Bwas 430 and increased to 1,038 prior to study entry. July 2014 inhibin Bhad stabilized to 1,106, August 1148, September 1053. Dose reduced forCycle 4 Day 15 to 750 mg/m² due to neutropenia (G3) despite interventionwith G-CSF. Completed study on this dose. End of Cycle 6 CT scan on11^(th) November confirmed Stable Disease to RECIST. Requestedcompassionate continuation. Due to diarrhoea following each dose,commenced Cycle 7 at the reduced dose of 625 mg/m². Developed emboliclose to Hickman line, and although this responded to clexane thepatient was withdrawn from study.

Stable Disease to RECIST (8 months).

Patient 050 (202) Oesophageal Cancer: Progressive Disease

Female (41 years)

Diagnosed with Stage 4 squamous cell carcinoma of the oesophagus withliver metastases in November 2013. Received 6 Cycles ofcisplatin+capecitabine from December 2013 until April 2014. Herpost-treatment scan showed progressive disease, and new lung deposits.

Commenced on NUC-1031 on 21^(st)May 2014 on 900 mg/m² weekly andreceived 3 Cycles. Following Cycle 1 Day 1 developed ALT and fatigue,both G3, and had dose reduction for Cycle 1 Day 8. End of Cycle 2 scanseemed to show Disease Progression, though uncertainty over somebaseline lesions.

As patient was deriving clinical benefit with improvement in herdysphagia, it was decided to allow one more cycle. End of Cycle 3 scanconfirmed Disease progression and patient was withdrawn from study.

Progressive Disease. Patient 051 (203) Anal Cancer: Progressive Disease

Female (51 years)

Diagnosed with metastatic squamous cell carcinoma of the anus in October2013. Progressed following 6 Cycles of cisplatin+5FU from October 2013until March 2014.

Commenced on NUC-1031 on 3 Jun. 2014 on 900 mg/m² weekly and hasreceived 2 Cycles. Required blood transfusion during Cycle 2 buttolerated treatment well. End of Cycle 2 CT scan showed progressivedisease.

Progressive Disease. Patient 052 (204) Oesophageal Cancer: StableDisease

Male (66 years)

Diagnosed with stage IV oesophageal cancer in December 2012. ReceivedFOX from January 2013 until July 2013, with a Partial Response. ShowedProgressive Disease in April 2014. Oesophageal stent inserted.

Commenced on NUC-1031 on 10 Jun. 2014 on 900 mg/m² weekly and completed1 Cycle. Due to fatigue dose was reduced to 825 mg/m² for Cycle 2, withgood effect. Completed 2 further Cycles at this dose. End of Cycle 2 CTscan showed Stable Disease to RECIST. Continues to have bone pain butscans revealed that this is not disease related. Dysphagia was becomingexacerbated. End of Cycle 4 CT scan on 10^(th) November revealed DiseaseProgression.

Stable Disease to RECIST (5 months).

Patient 053 (205) Colon Cancer: Progressive Disease

Female (31 years)

Diagnosed with a T3 N1 MO adenocarcinoma of the colon in 2008. Resectedand received 12 Cycles of FOLFOX, which was completed in 2009. Onprogression in 2011, received 13 cycles of FOLFIRI (6 of the cyclesincluded Avastin). With further progression in 2012, received 12 Cyclesof FOLFOX (6 of the Cycles included Avastin). Further ProgressiveDisease in January 2014, with metastases to the lungs and vertebrae.Received radiotherapy to the spine.

Commenced on NUC-1031 on 12 Jun. 2014 on 900 mg/m² weekly and hascompleted 1 Cycle. Has developed a series of infections, and hasrequired a de-functioning ileostomy. Following many treatment delaysreceived Cycle 2 Day 15 and end of Cycle 2 CT scan showed ProgressiveDisease with new lesions in the lung and liver.

Progressive Disease. Patient 055 (207) Ovarian Cancer: Stable Disease

Female (42 years)

Diagnosed in January 2002 with stage 2c grade 1 papillary serousadenocarcinoma of the ovary. Underwent, total abdominal hysterectomy,bilateral salpingo-oophorectomy and omentectomy with 6 Cycles ofadjuvant carboplatin+paclitaxel, completing treatment in June 2002.

In January 2012 developed a new grade 3 stage 3c primary peritonealcancer involving the recto-sigmoid junction. Underwent posteriorexenteration, comprising resection of caecum, rectum and sigmoid plusomentectomy and peritoneal stripping. From March to August 2012 received6 Cycles of adjuvant carboplatin+paclitaxel.

On disease recurrence in January 2013 received 6 Cycles of weeklypaclitaxel until May 2013. Following further progression in October 2013commenced 3 weekly paclitaxel+carboplatin+daily AKT inhibitor within theAKTRES study. In May 2014 CT showed new lesions and progressive disease.

Commenced on NUC-1031 on 2 Jul. 2014 on 900 mg/m² weekly and received 1Cycle. Dose was reduced to 825 mg/m² for Cycle 2 due to fatigue andcompleted 4 further Cycles. Experienced nausea and vomiting postchemotherapy, dose reduced to 625 mg/m² for Cycle 6 and completed onefurther cycle. End of Cycle 6 CT scan on 16 Dec. 2014 showed continuedStable Disease to RECIST with a reduction in tumour volume of 18% frombaseline. Patients CA 125 was 36 at baseline and was 24 in January 2015.Completed study and requested compassionate continuation. Received 2further Cycles under the Compassionate Access Programme with no furtherissues. Patient elected to come off study as she was traveling a greatdistance and requested the break.

Stable Disease to RECIST (10+ months).

Patient 057 (209) Ovarian Cancer: Stable Disease

Female (58 years)

Diagnosed with Stage 3c grade 3 serous ovarian cancer in October 2011.Received 3 Cycles of neo-adjuvant carboplatin+taxol, but developed taxolallergy on Cycle 3. Underwent posterior exenteration, ovarian debulking,anterior resection with a primary anastomosis, and omentectomy on 21Dec. 2011. Completed 3 Cycles of carboplatin+docetaxel in March 2012.Relapsed in June 2013 and received 6 Cycles of carboplatin+caelyx untilNovember 2013. In February 2014 had CT evidence of recurrent disease andreceived 3 Cycles of carboplatin+gemcitabine from March till June 2014.

Commenced on NUC-1031 on 9 Jul. 2014 on 900 mg/m² weekly and received 2doses. Presented for Cycle 1 Day 15 on 30^(th) July with anaemia andascites both G3.

Dose reduced to 825 for Cycle 2 and received 2 further Cycles. End ofCycle 4 CT scan showed continued Stable Disease to RECIST with areduction in tumour volume of 10% from baseline.Has a pleurx drain in and approximately 1,000 mls of very bloody fluidare withdrawn every other week. Further dose reduction to 750 mg/m² fromCycle 4 Day 1 due to fatigue, received one further cycle. Abdominalfluid increasing. Withdrawn from study due to clinical progression.Stable Disease to RECIST (5 months)

Patient 058 (210) Lung Cancer: Stable Disease

Male (54 years)

Diagnosed with metastatic non-small cell lung adenocarcinoma, with lymphnode and bone metastases in January 2014.

Commenced 3 Cycles of cisplatin and pemetrexed with a response of StableDisease. March 2014 commenced maintenance pemetrexed, received 4 Cyclesuntil April 2014. In May 2014 showed progressive disease with bilateralpulmonary metastases and received 1 more dose of pemetrexed.

Commenced on NUC-1031 on 14 Jul. 2014 on 825 mg/m² weekly and received 1Cycle. Cycle 1 Day 15 was delayed one week due to G3 transaminitis(ALT), a DLT, and dose was reduced to 750 mg/m². However some of theelevation in liver enzymes may be due to a congenital liver condition.Received 1 further Cycle at 750 mg/m². End of Cycle 4 CT scan showedProgressive Disease with new lesions in the bones.

Stable Disease to RECIST (4 months)

Patient 059 (211) Cervical Cancer: Stable Disease

Female (52 years)

Diagnosed with stage IV squamous cell carcinoma of cervix in December2012. Commenced on carboplatin+taxol chemotherapy, which was stopped dueto toxicity, especially from the taxol, (rash and itching), in February2013. Commenced 6 Cycles of cisplatin+topotecan from March 2013 untilJuly 2013 and achieved Stable Disease. CT scan in October 2013 showeddisease progression. She received 30Gy in 10 fractions pelvicradiotherapy, which was completed in November 2013. Commencedgemcitabine+carboplatin in December 2013 and received 3 Cycles. However,an interval CT scan in February 2014 showed disease progression.

Commenced on NUC-1031 on 24 Jul. 2014 on 825 mg/m²weekly and completed 1Cycle. Cycle 1 Day 15 was delayed for 1 week due to thrombocytopenia(G3). Dose reduced to 750 mg/m² for Cycle 2 and completed 2 furtherCycles. End of Cycle 4 CT scan showed continuing Stable Disease toRECIST. Dose reduced to 625 mg/m² for Cycle 4 due to fatigue (G3)experienced during Cycle 3 with good effect. Completed 2 further Cyclesat this dose. Urinary stents made patient very uncomfortable andreplaced in January 2015. During Cycle 6 patient reported to be verytired and elected to come off study.

Stable Disease to RECIST (7 months).

Patient 060 (212) Pancreatic Cancer: Progressive Disease

Male (83 years)

Diagnosed with metastatic pancreatic cancer (moderately differentiatedadenocarcinoma), with multiple liver metastases, in January 2014.Elected to have palliative chemotherapy on the Maestro study(gemcitabine on day 1, 8 and 15 and hypoxia activated TH302) fromJanuary to June 2014 but had progressive disease.

Commenced on NUC-1031 on 5 Aug. 2014 on 825 mg/m² weekly and completed 1Cycle. Presented on 10^(th) September with ALP, AST, ALT, all G3.Required new stent. Following treatment delays completed Cycle 2 on1^(st) October. CT scan showed Progressive Disease with new liverlesions.

Progressive Disease. Patient 061 (213) Colorectal Cancer: Stable Disease

Female (53 years)

Diagnosed with colon cancer in 2012. Surgery, involving bilateralsalpingo-oophorectomy, omentectomy and a loop ileostomy. Commenced 12Cycles of FOLFOX from January to July 2013. Following disease recurrencein April 2014 she received 8 Cycles of FOLFIRI and cetuximab from Aprilto July 2014. Had severe nausea and vomiting to all chemotherapy.

Commenced on NUC-1031 on 11 Aug. 2014 on 825 mg/m² weekly and completed4 Cycles. Had no nausea or vomiting during treatment. End of Cycle 2 CTscan showed Stable Disease to RECIST with a 2% reduction in tumourvolume. End of Cycle 4 CT scan showed Progressive Disease with newlesions in the spleen and liver.

Stable Disease to RECIST (3 months).

Patient 063 (215) Ovarian Cancer: Stable Disease

Female (78 years)

In May 2011 was diagnosed with concurrent vulval melanoma (Clark's level4) and a stage 3b ovarian cancer. Commenced 3 Cycles ofcarboplatin+paclitaxel from August to September 2011, followed byinterval de-bulking surgery, comprising total abdominal hysterectomy,bilateral salpingo-oophorectomy and omentectomy. She then received afurther 3 Cycles of carboplatin+paclitaxel, completing treatment inNovember 2011. On disease progression, commenced 6 Cycles ofgemcitabine+carboplatin+Avastin from September 2013 to February 2014.Then received 2 Cycles of caelix from April to June 2014.

Commenced on NUC-1031 on 27 Aug. 2014 on 825 mg/m² weekly and received 1Cycle. Dose reduced to 750 mg/m² for Cycle 2 Day 15 due to anaemia andneutropenia and received 2 further Cycles. End of Cycle 2 CT scan showedStable Disease to RECIST. Dose reduced to 625 mg/m² for Cycle 4 Day 1due to fatigue (G3), experienced on Cycle 3. Developed persistentshortness of breath. Removed from study.

Stable Disease to RECIST (3 months).

Patient 064 (216) Trophoblastic Cancer: Progressive Disease

Female (38 years)

In June 2011 was diagnosed with recurrent stage 3 mixed placental siteand epithelioid trophoblastic tumour (PSTT/ETT). Following radicalhysterectomy and lymph node sampling received adjuvant chemotherapy withpaclitaxel+cisplatin ultimately with paclitaxel+etoposide from June toOctober 2011 followed by surgery for lymph leakage in November 2011 anda bilateral ureteric implantation February 2012.

In February 2013 underwent a left oophorectomy and resection of bladderserosa for recurrent disease and was given adjuvant chemotherapy of highdose etoposide from March to July 2013, followed by autologous stem celltransplant. January 2014 underwent posterior exenteration, comprisingresection of the upper vagina, rectum and bladder, removal of the leftovary, anterior vagina, a mesenteric nodule and right ureter. Received 5Cycles of pemetrexed+carboplatin from February to May 2014. This wasswitched to gemcitabine+carboplatin, due to rising HCG levels. Received2 Cycles but interval CT scan showed left lower lobe lung lesion whichwas considered inoperable.

Commenced on NUC-1031 on 3 Sep. 2014 on 825 mg/m² weekly and hasreceived 2 Cycles. End of Cycle 2 CT scan showed Progressive Disease,growth in existing lesions in lungs and peritoneum.

Progressive Disease. Patient 066 (218) Colorectal Cancer: Stable Disease

Male (65 years)

In May 2011 was first diagnosed with pT4b pN0 moderately differentiatedadenocarcinoma of the sigmoid colon. Underwent an anterior resection inNovember 2011 and commenced FOLFOX chemotherapy in January 2012.Developed peripheral neuropathy following 3 Cycles and was switched to5FU monotherapy. Also troubled with delays due to diarrhoea and malaise.Following 3 months on 5FU showed a stable marker response but a mixedresponse on CT scan in July 2012. Remained stable off treatment untilMay 2013 showed progressing on his CT scan with the tumour markerslevels doubling. Re-commenced 5FU+Avastin, which was completed inOctober 2013. Treatment was complicated with numerous hospitaladmissions with chest infections and chest pain. Received 8 Cycles ofcetuximab from March to July 2014. CT scan showed disease progressionand switched to 5FU plus Avastin. In June 2014 underwent a laparotomyand adhesiolysis because of adhesions from metastatic deposits withinhis peritoneum. Received a further cycle of Avastin but, with a risingCEA, was discontinued. Has many co morbidities, COPD, ischemic heartdisease, and congestive cardiac failure.

Commenced on NUC-1031 on 16 Sep. 2014 on 825 mg/m² weekly and received 2Cycles. Dose reduction to 750 mg/m² for Cycle 3 due to fatigue andreceived 1 further Cycle. End of Cycle 2 CT scan showed Stable Diseaseto RECIST.

Had two recent admissions for complications from a hernia, whichresulted in treatment delays. Unscheduled CT scan on 30^(th) Decembershowed continued Stable Disease with reduction in tumour volume of 16%from baseline. Dose further reduced to 625 mg/m² for Cycle 4 due tofatigue. Has received 1 further Cycle at this dose with no furtherissues. Admitted during Cycle 5 with acute back pain, old fracture noted(not study related). Continues under surgical evaluation. Withdrawn fromstudy due to treatment delays.Stable Disease to RECIST (8+ months).

Patient 067 (219) Osteosarcoma: Stable Disease

Male (38 years)

In May 2011 was diagnosed 24 Feb. 2012 with osteosarcoma of proximalright tibia. Received 6 Cycles of cisplatin+doxorubicin+methotrexatefrom February to November 2012. Had a proximal tibial replacement onNovember 2012 and received post chemotherapy mifamurtide for 6 months.CT scan in August 2014 showed metastatic recurrence with newintrapulmonary and pericardial lesion, inferior to IVC and adjacent toright atrium.

Commenced on NUC-1031 on 30 Sep. 2014 on 825 mg/m² weekly and hasreceived 5 Cycles. End of Cycle 4 CT scan showed Stable Disease toRECIST. Tends to develop neutropenia G2 towards the end of each Cyclebut counts bounce back quickly. Completed study on 3 Mar. 2015.Tolerated study drug well. EOS CT scan showed Stable Disease to RECISTwith an increase in 1% from baseline. Scan also showed significantcalcification to tumour. Patient requested compassionate continuation ofstudy drug and will completed C7 on 2 Apr. 2015 at the reduced dose of750 mg/m2. Assessed by the thoracic surgeon who will operate on the leftlung to remove the target lesion on the lower lobe on 26 Apr. 2015.Thoracic surgeons removed the calcified target lesion in the lower lobeon 26 Apr. 2015. The lesion was removed completely with a clear marginof normal surrounding tissue. The patient has made a good recovery fromthe operation.

Stable Disease to RECIST (7+ months).

Patient 068 (220) Lung Cancer

Male (60 years)

In May 2011 was diagnosed with T3 N3 M1b non-small cell carcinoma of theright lung (adenocarcinoma), EGFR wild type, in February 2013. Received10 Cycles of pemetrexed and cisplatin from February to December 2013,followed by thoracic palliative radiotherapy. From January to May 2014enrolled in the POPLAR study on the docetaxel arm.

Commenced on NUC-1031 on 3 Oct. 2014 on 825 mg/m² weekly and received 2Cycles. End of Cycle 2 CT scan showed Progressive Disease. Removed fromstudy.

Progressive Disease. Patient 069 (221) Colorectal Cancer

Female (45 years)

In May 2011 was diagnosed with colorectal cancer. In August 2011received neo-adjuvant capecitabine with radiotherapy and then primarydebulking surgery in December 2011. Received adjuvant FOLFOX fromJanuary to July 2012. In April 2013 developed a solitary lungrecurrence, which was resected. July 2013, a right parieto-occipitalrecurrence was found and removed, along with some dermal andsubcutaneous cancer deposits. Commenced on cetuximab with FOLFIRI fromSeptember 2013 and remained on maintenance cetuximab until September2014. Gamma-knife treatment in September 2014 for brain metastasis. Isasymptomatic for neurological symptoms.

Commenced on NUC-1031 on 9 Oct. 2014 on 825 mg/m² weekly and received 3Cycles. PET scan on 30/10 showed Partial Response. During pre C2examination cutaneous and sub cutaneous metastases were greatly reducedor almost vanished and no new ones have appeared. End of C2 CT scanshowed Stable Disease to RECIST with an 11% reduction in tumour volumefrom baseline. Developed neutropenia (G4) and leukopenia (G3) duringCycle 3 and dose reduced to 750 mg/m² for Cycle 4. End of C4 CT scanshowed Stable Disease to RECIST with 26% reduction in tumour volume frombaseline. Following Cycle 4 D1 developed neutropenia and leukopenia, G3and was dose reduced to 675 mg/m² for Cycle 4 D8. Dose was reduced to625 mg/m² for C5 D8 due to neutropenia and leukopenia, G2. Experiencingvisual disturbances, CT scan showed lesion in brain had increased. Thishas been removed with cyberknife. She will recommence on study drug, C7D1 on 29 Apr. 2015 at the reduced dose of 500 mg/m².

Stable Disease to RECIST (7+ months).

We claim:
 1. A method of killing cancer stem cells in a patient withcancer, comprising administering to the patient in need thereof aneffective amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof in combination with achemotherapeutic agent, wherein the cancer is selected from the groupconsisting of lung cancer, liver cancer, breast cancer, head and neckcancer, neuroblastoma, thyroid carcinoma, skin cancer, oral squamouscell carcinoma, urinary bladder cancer, pancreatic cancer, colon cancer,colorectal cancer and gynecological cancer.
 2. The method of claim 1,wherein the chemotherapeutic agent is an inhibitor of the VEGF pathway.3. The method of claim 2, wherein the inhibitor of the VEGF pathway isbevacizumab.
 4. The method of claim 1, wherein the chemotherapeuticagent is a mitotic inhibitor.
 5. The method of claim 1, wherein thechemotherapeutic agent is a monoclonal antibody.
 6. The method of claim5, wherein the monoclonal antibody is a passive or active immunotherapy.7. The method of claim 1, wherein the chemotherapeutic agent is atopoisomerase inhibitor.
 8. The method of claim 1, wherein thechemotherapeutic agent is a metallic agent.
 9. The method of claim 1,wherein the cancer is relapsed or refractory.
 10. The method of claim 9,wherein the cancer is relapsed.
 11. The method of claim 9, wherein thecancer is refractory.
 12. The method according to claim 1, wherein thecompound is administered intravenously.
 13. The method of claim 1wherein the cancer is colorectal cancer.
 14. The method of claim 1,wherein the cancer is lung cancer.
 15. The method of claim 1, whereinthe compound and chemotherapeutic agent are administered at the sametime.
 16. The method of claim 1, wherein the compound andchemotherapeutic agent are administered at the different times.
 17. Themethod of claim 16, wherein the compound is administered prior to thechemotherapeutic agent.
 18. The method of claim 15, wherein the compoundis administered after the chemotherapeutic agent.